Antigenic epstein barr virus polypeptides

ABSTRACT

This disclosure relates to antigenic EBV polypeptides and their use in eliciting antibodies against EBV. Also disclosed are antigenic polypeptides comprising an EBV polypeptide and a ferritin protein.

This application is a continuation of U.S. application Ser. No.17/061,146, filed Oct. 1, 2020, which is a continuation of InternationalApplication No. PCT/US2019/025419, filed Apr. 2, 2019, which claims thebenefit of priority of U.S. Provisional Patent Application No.62/652,201, filed Apr. 3, 2018, the entire contents of which areincorporated herein by reference.

The present application contains a Sequence Listing which has beensubmitted electronically in XML format. Said XML copy, created on Nov.28, 2022, is named 2022-11-28_01121-0032-01US_SL_ST26.txt and is 178,732bytes in size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

Even with many successes in the field of vaccinology, new breakthroughsare needed to protect humans against many life-threatening infectiousdiseases. Many currently licensed vaccines rely on decade-oldtechnologies to produce live-attenuated or inactivated killed pathogens,which carry inherent safety concerns and in many cases, stimulate onlyshort-lived, weak immune responses that require the administration ofmultiple doses. While advances in genetic and biochemical engineeringhave made it possible to develop therapeutic agents to challengingdisease targets, these applications to the field of vaccinology have notbeen fully realized. Recombinant protein technologies now allow thedesign of optimal antigens. Additionally, nanoparticles haveincreasingly demonstrated the potential for optimal antigen presentationand targeted drug delivery. Nanoparticles with multiple attachedantigens have been shown to have increased binding avidity afforded bythe multivalent display of their molecular cargos, and an ability tocross biological barriers more efficiently due to their nanoscopic size.Helicobacter pylori (H. pylori) ferritin nanoparticles fused toinfluenza virus haemagglutinin (HA) protein has allowed improved antigenstability and increased immunogenicity in mouse influenza models (seeKanekiyo et al., Nature 499:102-106 (2013)). This fusion proteinself-assembled into an octahedrally-symmetric nanoparticle and presented8 trimeric HA spikes to give a robust immune response in variouspre-clinical models when used with an adjuvant.

Epstein Barr virus (EBV) infects about 95% of the adult populationworldwide and has been known to be associated with two B-cell lymphomas,Burkitt's and Hodgkin's lymphomas. The virus can also infect epithelialcells and is associated with nasopharyngeal cancer. Furthermore, EBVcauses most cases of infectious mononucleosis in developed countries,affecting mainly children and young adults. Infectious mononucleosis canresult in a long recovery period of up to one month. There are currentlyno approved vaccines on the market, so there is a strong need for apreventive vaccine.

Here, a set of new polypeptides, nanoparticles, compositions, methods,and uses involving EBV polypeptides is presented. Novel EBV single-chaingL and gH (sometimes depicted as gL/gH or gH/gL) polypeptides weregenerated, as were antigenic polypeptides comprising these novel EBVpolypeptides and ferritin. Antigenic polypeptides and nanoparticlescomprising the single-chain gL and gH polypeptides can comprise arelatively long linker between the gL and gH sequences, which wasobserved to provide an increase in immunogenicity. Antigenic ferritinpolypeptides and nanoparticles comprising EBV gp220 polypeptides werealso generated. Furthermore, self-adjuvanting antigenic polypeptidescomprising the described EBV polypeptides and ferritin were developedwherein immune-stimulatory moieties, such as adjuvants, were directly,chemically attached to the antigenic polypeptide. The direct conjugationof an immune-stimulatory moiety to the antigenic polypeptide allows fortargeted co-delivery of the immune-stimulatory moiety and EBVpolypeptide in a single macromolecular entity, which can greatlydecrease the potential for systemic toxicity that is feared withtraditional vaccines that comprise antigens and immune-stimulatorymolecules such as adjuvants as separate molecules. The co-delivery ofimmune-stimulatory moieties together with EBV polypeptides in amacromolecular entity and their multivalent presentation may also reducethe overall dose needed to elicit protection, reducing manufacturingburdens and costs.

SUMMARY

It is an object of this disclosure to provide compositions, kits,methods, and uses that can provide one or more of the advantagesdiscussed above, or at least provide the public with a useful choice.Accordingly, the following embodiments are disclosed herein.

Embodiment 1 is an antigenic EBV polypeptide comprising an Epstein BarrVirus (EBV) gL polypeptide and an EBV gH polypeptide, wherein a linkerhaving a length of at least 15 amino acids separates the EBV gLpolypeptide and the EBV gH polypeptide.

Embodiment 2 is an antigenic EBV polypeptide comprising an Epstein BarrVirus (EBV) gL polypeptide, an EBV gH polypeptide, and an EBV gp42polypeptide, wherein a linker having a length of at least 15 amino acidsseparates the EBV gL polypeptide and the EBV gH polypeptide.

Embodiment 3 is the antigenic EBV polypeptide of embodiment 1 orembodiment 2, further comprising a ferritin.

Embodiment 4 is an antigenic EBV polypeptide comprising an EBVpolypeptide and a ferritin protein, wherein the ferritin proteincomprises a mutation replacing a surface-exposed amino acid with acysteine.

Embodiment 5 is the antigenic EBV polypeptide of embodiment 4, whereinthe EBV polypeptide comprises an EBV gL polypeptide, an EBV gHpolypeptide, or an EBV gp220 polypeptide.

Embodiment 6 is the antigenic EBV polypeptide of embodiment 5, whereinthe EBV polypeptide comprises a gL polypeptide and the polypeptidefurther comprises an EBV gH polypeptide.

Embodiment 7 is the antigenic EBV polypeptide of any one of embodiments1 or 3-6, wherein the polypeptide further comprises an EBV gp42polypeptide.

Embodiment 8 is a composition comprising a first antigenic EBVpolypeptide and a second antigenic EBV polypeptide, wherein the firstantigenic EBV polypeptide comprises a ferritin heavy chain and a firstEBV polypeptide, the second antigenic EBV polypeptide comprises aferritin light chain and a second EBV polypeptide, and the first andsecond EBV polypeptides are different.

Embodiment 9 is the composition of embodiment 8, wherein the first EBVpolypeptide or the second EBV polypeptide comprises a gp220 polypeptide.

Embodiment 10 is the composition of any one of embodiments 8 to 9,wherein (i) the first antigenic EBV polypeptide comprises one or both ofa gL polypeptide and a gH polypeptide and the second antigenic EBVpolypeptide comprises a gp220 polypeptide, or (ii) the first antigenicEBV polypeptide comprises a gp220 polypeptide and the second antigenicEBV polypeptide comprises one or both of a gL polypeptide and a gHpolypeptide.

Embodiment 11 is the composition of any one of embodiments 8 to 10,wherein the first antigenic EBV polypeptide comprises a gL polypeptideand a gH polypeptide; or the second antigenic EBV polypeptide comprisesa gL polypeptide and an EBV gH polypeptide.

Embodiment 12 is the composition of embodiment 10 or 11, wherein theantigenic EBV polypeptide comprising a gL polypeptide and/or a gHpolypeptide further comprises a gp42 polypeptide.

Embodiment 13 is the antigenic EBV polypeptide or composition of any oneof embodiments 1-12, comprising a gH and gL polypeptide, wherein the gHpolypeptide is C-terminal to the gL polypeptide, optionally comprising agp42 polypeptide, wherein the gp42 polypeptide is C-terminal to the gHpolypeptide.

Embodiment 14 is the antigenic EBV polypeptide or composition of any oneof embodiments 1-13, comprising a gp42 polypeptide, wherein the gp42polypeptide comprises an amino acid sequence with at least 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 239 or 240.

Embodiment 15 is the antigenic EBV polypeptide or composition of any oneof embodiments 1-14, comprising an EBV gH polypeptide and an EBV gp42polypeptide, wherein a linker having a length of at least 15 amino acidsseparates the EBV gH polypeptide and the EBV gp42 polypeptide,optionally wherein the linker has a length of 15 to 60 amino acids, 20to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to 50amino acids, or 40 to 50 amino acids, further optionally wherein thelinker comprises an amino acid sequence with at least 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 234.

Embodiment 16 is the antigenic EBV polypeptide or composition of any oneof the preceding embodiments, comprising a linker, wherein the linkerhas a length of at least 15 amino acids, optionally wherein the linkerseparates a first EBV polypeptide and a second EBV polypeptide.

Embodiment 17 is the antigenic EBV polypeptide or composition ofembodiment 16, wherein the linker has a length of 15 to 60 amino acids,20 to 60 amino acids, 30 to 60 amino acids, 40 to 60 amino acids, 30 to50 amino acids, or 40 to 50 amino acids.

Embodiment 18 is the antigenic EBV polypeptide or composition of any oneof the preceding embodiments, wherein the EBV polypeptide comprises anamino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% identity to SEQ ID NO: 36.

Embodiment 19 is the antigenic EBV polypeptide or composition of any oneof the preceding embodiments, wherein the EBV polypeptide comprises anamino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% identity to SEQ ID NO: 37.

Embodiment 20 is the antigenic EBV polypeptide or composition of any oneof the preceding embodiments, wherein the polypeptide comprises a linkercomprising an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%,98%, 99%, or 100% identity to SEQ ID NO: 30, optionally wherein thelinker separates a first EBV polypeptide and a second EBV polypeptide.

Embodiment 21 is the antigenic EBV polypeptide or composition of any oneof the preceding embodiments, wherein the EBV polypeptide comprises anamino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% identity to SEQ ID NO: 38.

Embodiment 22 is the antigenic EBV polypeptide or composition of any oneof embodiments 3-21, further comprising a further linker that separatesthe EBV polypeptide and the ferritin.

Embodiment 23 is the antigenic EBV polypeptide or composition of any oneof embodiments 3-22, comprising an EBV gp42 polypeptide locatedN-terminal to the ferritin and C-terminal to the gH polypeptide, whereina linker separates the EBV gp42 polypeptide and the ferritin, optionallywherein the linker has a length of at least 15 amino acids or has alength of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60 aminoacids, 40 to 60 amino acids, 30 to 50 amino acids, or 40 to 50 aminoacids, further optionally wherein the linker comprises an amino acidsequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to any one of SEQ ID NOs: 233, 234, 235, 236, 237, or 238.

Embodiment 24 is the antigenic EBV polypeptide or composition ofembodiments 22 or 23, wherein the linker comprises a cysteine.

Embodiment 25 is the antigenic EBV polypeptide or composition of any oneof embodiments 22-24, wherein the linker comprises an amino acidsequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 33.

Embodiment 26 is the antigenic EBV polypeptide or composition ofembodiments 24-25, wherein the cysteine is conjugated to animmune-stimulatory moiety, optionally wherein the immune-stimulatorymoiety is an agonist of TLR2, TLR7/8, TLR9, or STING.

Embodiment 27 is the antigenic EBV polypeptide or composition of any oneof embodiments 3-26, wherein the ferritin comprises one or more of E12C,S26C, S72C, A75C, K79C, S100C, and S111C mutations of H. pylori ferritinor one or more corresponding mutations in a non-H. pylori ferritin asdetermined by pairwise or structural alignment.

Embodiment 28 is the antigenic EBV polypeptide or composition of any oneof embodiments 3-27, wherein the ferritin comprises a mutation replacinga surface-exposed asparagine with a non-asparagine amino acid,optionally wherein the asparagine is at position 19 of H. pyloriferritin, or an analogous position in a non-H. pylori ferritin asdetermined by pairwise or structural alignment.

Embodiment 29 is the antigenic EBV polypeptide or composition of any oneof embodiments 3-28, wherein the ferritin comprises a mutation replacingan internal cysteine with a non-cysteine amino acid, optionally whereinthe internal cysteine is at position 31 of H. pylori ferritin, or aposition that corresponds to position 31 of H. pylori ferritin asdetermined by pair-wise or structural alignment.

Embodiment 30 is the antigenic EBV polypeptide or composition of any oneof embodiments 3-29, wherein the ferritin comprises an amino acidsequence with 80%, 85%, 90%, 95%, 98%, or 99% identity to any one of SEQID NOs: 201-207 or 211-215.

Embodiment 31 is the antigenic EBV polypeptide or composition of any oneof embodiments 1-31, wherein the antigenic EBV polypeptide comprises asequence with at least 80%, 85%, 90%, 95%, 98%, or 99% identity to aminoacids 23-1078 of SEQ ID NO: 226.

Embodiment 32 is the antigenic EBV polypeptide or composition of any oneof embodiments 1-31, wherein the antigenic EBV polypeptide comprises asequence with at least 80%, 85%, 90%, 95%, 98%, or 99% identity to anyone of SEQ ID NOs: 226-231 or 241-242, optionally lacking the leadersequence.

Embodiment 33 is a ferritin particle comprising the antigenic EBVpolypeptide or the first and second polypeptides of any one ofembodiments 3-32.

Embodiment 34 is a composition comprising the antigenic EBVpolypeptide(s) or ferritin particle of any one of the precedingembodiments and a pharmaceutically acceptable carrier.

Embodiment 35 is the composition of embodiment 34, wherein the ferritinparticle comprises an EBV gL polypeptide and an EBV gH polypeptide, andthe composition further comprises a second ferritin particle comprisinga gp220 polypeptide.

Embodiment 36 is the antigenic EBV polypeptide, ferritin particle, orcomposition of any one of the preceding embodiments for use in a methodof eliciting an immune response to influenza or in protecting a subjectagainst infection with EBV.

Embodiment 37 is a method of eliciting an immune response to EBV orprotecting a subject against infection with EBV comprising administeringany one or more antigenic EBV polypeptide, ferritin particle, orcomposition of any one of the preceding embodiments to a subject.

Embodiment 38 is the antigenic EBV polypeptide, ferritin particle,composition, or method of any one of embodiments 36-37, wherein thesubject is human.

Embodiment 39 is a nucleic acid encoding the antigenic EBV polypeptideof any one of embodiments 1-32, optionally wherein the nucleic acid isan mRNA.

Additional objects and advantages will be set forth in the descriptionwhich follows, and/or will be obvious from the description, or may belearned by practice. The objects and advantages will be realized andattained by means of the elements and combinations particularly pointedout in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate certain embodiments and togetherwith the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show purified single-chain gL and gH monomer (FIG. 1A) (SEQID NO: 6) and trimer (FIG. 1B) (SEQ ID NO: 11) with and without removalof the His-tag by Coomassie and Western blot analysis. FIG. 1B alsopresents the UV absorbance trace of fractions from a size exclusioncolumn (Superose® 6) purification.

FIGS. 2A-2E shows purification and characterization of single-chaingL/gH-ferritin nanoparticles (SEQ ID NO: 14). A UV absorbance trace ofSuperose® 6 purification fractions is shown (FIG. 2A), as well asCoomassie (FIG. 2B) and Western blot (FIG. 2C) analysis of selectedfractions from the purification (L indicates molecular weight ladder;the positions of the 150 and 250 kDa bands are indicated at right inFIG. 2B). Dynamic light scattering (FIG. 2D) and electron microscopy(FIG. 2E) analyses of the single-chain nanoparticles are also presented.

FIG. 3 shows different representative single-chain gL/gH-ferritinconstructs.

FIG. 4 shows antibody titers following immunization of mice withsingle-chain gL/gH trimers or nanoparticles (NP) admixed with AF03adjuvant, which is a squalene emulsion-based adjuvant. *p-value=<0.05when comparing the NP construct with its corresponding trimer construct.From left to right, constructs were SEQ ID NOs: 16, 10, 11, 13, 12, and14.

FIGS. 5A-5B show anti-gL/gH antibody response in mice to a bivalentcomposition comprising both gp220 nanoparticles (SEQ ID NO: 1) and asingle-chain gL/gH nanoparticle (“gL_gH_C5 NP,” SEQ ID NO: 19) comparedto the single-chain gL/gH nanoparticle and negative control nakedferritin (i.e., ferritin not conjugated to any non-ferritin polypeptideor immune-stimulatory moiety). The results indicate that using thebivalent composition did not result in interference with the anti-gL/gHantibody response relative to the results with single-chain gL/gH withnegative control naked ferritin. Both compositions included AF03adjuvant. ELISA results at individual dilutions (FIG. 5A) and bindingtiter (FIG. 5B) are shown.

FIGS. 6A-6B show anti-gp220 antibody response to a bivalent compositioncomprising both gp220 nanoparticles and single-chain gL/gH nanoparticlesas described for FIGS. 5A-B. The results indicate that using thebivalent composition did not result in interference with the anti-gp220antibody response relative to the results with gp220 nanoparticles withnegative control naked ferritin. Both compositions included AF03adjuvant. ELISA results at individual dilutions (FIG. 6A) and bindingtiter (FIG. 6B) are shown.

FIG. 7A shows the design of a nanoparticle comprising an EBV polypeptideand ferritin comprising a mutation replacing a surface-exposed aminoacid with a cysteine for conjugation to an immune-stimulatory moietysuch as a toll-like receptor (TLR) agonist. For an exemplary sequencecorresponding to this design, see SEQ ID NO: 14, in which a single-chaingL/gH antigen is linked to ferritin by a flexible 46 amino acid linker.FIG. 7B shows a representative toll-like receptor (TLR) agonist (SM7/8awith a PEG4-maleimide linker) suitable for conjugation to a constructaccording to FIG. 7A. FIG. 7C shows an electron micrograph (EM) image ofa gL/gH nanoparticle with SM7/8a conjugated thereto via the cysteine onthe ferritin surface and a PEG4-maleimide linker.

FIG. 8A shows a structure of part of a ferritin comprising a mutationreplacing a surface-exposed amino acid with a cysteine, in which thelocation of the cysteine is indicated. FIG. 8B illustrates conjugationof a CpG adjuvant (SEQ ID NO: 247) to ferritin by juxtaposing theferritin, linker, and CpG adjuvant, oriented to show the parts of eachmoiety that become attached to each other in proximity.

FIGS. 9A-9B show mass spectrometry (MS) spectra of the unconjugated(FIG. 9A) and SM7/8a-conjugated (FIG. 9B) forms of a gL/gH-ferritin. Thedifference in mass of the main peaks was 711 Da, which approximatelycorresponds to the predicted difference from conjugating the SM7/8a withlinker.

FIGS. 10A-10B show mass spectrometry (MS) spectra of the unconjugated(FIG. 10A) and SM7/8a-conjugated (FIG. 10B) forms of a gp220-ferritin.The difference in mass of the main peaks was 714.7 Da, whichapproximately corresponds to the predicted difference from conjugatingthe SM7/8a with linker.

FIGS. 11A-11D show electron microscopy (EM) images of unconjugated(FIGS. 11A, C) and conjugated (FIGS. 11B, D) single-chain gL/gH (FIGS.11A, B) and gp220 (FIGS. 11C, D) ferritin nanoparticles, indicating thatconjugation of SM7/8a to these nanoparticles did not disruptnanoparticle structure.

FIGS. 12A-12B show antibody responses in mice after treatment withferritin nanoparticles comprising single-chain gL/gH either withoutconjugated SM7/8a or other adjuvant, with AF03 adjuvant as a separatemolecule, or with conjugated SM7/8a. ELISA results are shown asindividual dilutions (FIG. 12A) and binding titers (FIG. 12B).

FIGS. 13A-13B show antibody responses in mice after treatment withnanoparticles comprising gp220 either alone, with AF03 adjuvant as aseparate molecule, or with conjugated SM7/8a. ELISA results are shown asindividual dilutions (FIG. 13A) and binding titers (FIG. 13B).

FIGS. 14A-14B show anti-gL/gH antibody responses in mice after treatmentwith gp220 nanoparticles conjugated to SM7/8a and single-chain gL/gHferritin nanoparticles conjugated to SM7/8a compared to treatment withsingle-chain gL/gH ferritin nanoparticles conjugated to SM7/8a and nakedferritin, as measured by ELISA. Results are shown for experimentswithout (FIG. 14A) or with (FIG. 14B) admixed AF03.

FIGS. 15A-15B show anti-gp220 antibody responses in mice after treatmentwith gp220 nanoparticles conjugated to SM7/8a and single-chain gL/gHnanoparticles conjugated to SM7/8a compared to treatment with gp220nanoparticles conjugated to SM7/8a and naked ferritin, as measured byELISA. Results are shown for experiments without (FIG. 15A) or with(FIG. 15B) admixed AF03.

FIG. 16 shows anti-gL/gH antibody responses in mice after treatment withsingle-chain gL/gH nanoparticles (gL/gH_C5; SEQ ID NO: 19) and nakedferritin, with or without either or both of admixed AF03 adjuvant and/orSM7/8a conjugated to the single-chain gL/gH nanoparticles, as measuredby ELISA endpoint titer.

FIG. 17 shows anti-gp220 antibody responses in mice after treatment withgp220 nanoparticles and naked ferritin, with or without either or bothof admixed AF03 adjuvant and/or SM7/8a conjugated to the gp220nanoparticles.

FIG. 18 shows anti-gL/gH antibody responses in mice after treatment withbivalent compositions comprising gp220 nanoparticles and single-chaingL/gH nanoparticles. As indicated in the legend, some nanoparticles wereconjugated to SM7/8a, and some nanoparticles were admixed with AF03. Theorder of symbols from top to bottom in the key matches the order ofsymbols from left to right in the graph.

FIG. 19 shows anti-gp220 antibody responses in mice after treatment withbivalent compositions comprising single-chain gL/gH nanoparticles andgp220 nanoparticles and/or naked ferritin. As indicated in the legend,some nanoparticles were conjugated to SM7/8a, and some nanoparticleswere admixed with AF03. The order of symbols from top to bottom in thekey matches the order of symbols from left to right in the graph.

FIGS. 20A-20D show antibody responses in mice after treatment withgL_gH_C7 nanoparticle (SEQ ID NO: 20) with or without admixed AF03adjuvant and/or conjugation to SM7/8a (FIG. 20A, 20C, or 20D) or CpGoligodeoxynucleotide (FIG. 20B). Shown are endpoint titer from primebleed measured by ELISA (FIG. 20A), ELISA results at individualdilutions from booster bleed (FIG. 20B), and endpoint titers measured byELISA from booster bleed (FIG. 20C) and terminal bleed (FIG. 20D).

FIG. 21 shows antibody responses in mice after treatment with thegL_gH_C5 nanoparticle (SEQ ID NO: 19) with or without admixed AF03adjuvant and/or conjugation to SM7/8a as endpoint titers measured byELISA from the prime, boost, and terminal bleeds.

FIG. 22 shows antibody responses in mice after treatment with a gp220nanoparticle with or without admixed AF03 adjuvant and/or conjugation toSM7/8a as endpoint titers measured by ELISA from the prime, boost, andterminal bleeds.

FIG. 23A shows the light and heavy chains of T. ni ferritin with orwithout fusion to either gp220 or gL/gH, visualized by Coomassiestaining (FIG. 23A). The 20, 25, 75, 100, and 150 kDa markers in therightmost lane of FIG. 23A are labeled. FIG. 23B provides anillustration of the constructs comprising light and heavy chains of T.ni ferritin with or without fusion to either gp220 or gL/gH.

FIGS. 24A-24D demonstrate ion exchange (Q column) chromatographic andsize-exclusion chromatographic (SEC) purification of gp220-T. niferritin. Shown are absorbance traces from the Q column at pH 7 (FIG.24A) and SEC Superose® 6, 16/600 (FIG. 24B); Coomassie staining offractions from Q column at pH 7 (FIG. 24C) (lanes from left are input(“In”), flow-through (“FT”), mol. wt. ladder (sizes labeled at left inkD), and selected fractions); and Coomassie staining of fractions fromSEC Superose® 6, 16/600 (FIG. 24D) (lanes from left are mol. wt. ladder(sizes labeled at left in kD) and selected fractions). FIG. 24E shows anillustration of the constructs.

FIGS. 25A-25D demonstrate ion exchange (Q column) chromatographic andsize-exclusion chromatographic (SEC) purification of gL/gH (Light)/gp220(Heavy)-T. ni ferritin. Shown are absorbance traces from the Q column atpH 7 (FIG. 25A) and SEC Superose® 6, 16/600 (FIG. 25B); Coomassiestaining of fractions from Q column at pH 7 (FIG. 25C) (lanes from leftare input (“In”), flow-through (“FT”), mol. wt. ladder (sizes labeled atleft in kD), and selected fractions); and Coomassie staining offractions from SEC Superose® 6, 16/600 (FIG. 25D) (lanes from left aremol. wt. ladder (sizes labeled at left in kD) and selected fractions).FIG. 25E shows an illustration of the constructs.

FIGS. 26A-26H show gp220-T. ni ferritin or gL/gH (lightchain)/gp220(heavy chain)-T. ni ferritin constructs visualized byCoomassie staining Coomassie (FIG. 26A and FIG. 26E), illustrateddiagrammatically (FIG. 26B and FIG. 26F), characterized by dynamic lightscattering (DLS) (FIG. 26D and FIG. 26H), and visualized in electronmicrographs (FIG. 26C and FIG. 26G). FIGS. 26A-26D present data withgp220 fused to both the light and the heavy chain (diagram in FIG. 26B).FIGS. 26E-26H present data with gp220 fused to the heavy chain and gL/gHfused to the light chain (diagram in FIG. 26F).

FIGS. 27A-FIG. 27C show naked T. ni ferritin particles (i.e., not fusedto a non-ferritin polypeptide) visualized by Coomassie staining (FIG.27A), visualized in electron micrographs (FIG. 27B), and characterizedby DLS (FIG. 27C).

FIG. 28A shows a SDS denaturing Coomassie-stained gel (at left) with apurified gH/gL/gp42_NP construct (SEQ ID NO: 227), which was expressedin 293 expi cells, and (at right) the size exclusion chromatography(SEC) peak of the gH/gL/gp42_NP. Units of the horizontal axis of the SECchromatogram are mL. FIG. 28B shows the gH/gL/gp42_NP purified from theCHO pools to have a dynamic light scattering radius of around 26.2 nm.

FIG. 29A-B show an assessment of the immune response elicited by amonovalent gH/gL/gp42 nanoparticle composition in combination with anaked ferritin nanoparticle or a bivalent composition (gH/gL/gp42nanoparticle in combination with a gp220). FIG. 29A shows B cellneutralization. FIG. 29B shows epithelial cell neutralization.

FIGS. 30A-E show endpoint binding titers against the indicated antigens.FIGS. 30F-G show an EBV viral neutralizing assay (in B cells andepithelial cells, respectively) of sera from ferrets vaccinated asindicated. Prime=Inj. 1 and Boost=Inj. 2.

FIGS. 31A-B: FIG. 31A shows purification of gH/gL/gp42_NP_C12 (SEQ IDNO: 228) using Superose 6 size exclusion chromatography. The arrowdepicts the fractions collected from the peak with a denaturingcoomassie gel analysis and a western blot analysis using anti-ferritinantibodies. FIG. 31B is a dynamic light scattering analysis of thesample in FIG. 31A, which shows the particle size radius of 20.6 nm.

FIGS. 32A-B: FIG. 32A shows purification of gH/gL/gp42_NP_C13 (SEQ IDNO: 229) using the Superose 6 size exclusion chromatography. The arrowdepicts the fractions collected from the peak with a denaturingcoomassie gel analysis and a western blot analysis using anti-ferritinantibodies. FIG. 32B is a dynamic light scattering analysis of thesample in FIG. 32A, which shows the particle size radius of 17.1 nm.

FIGS. 33A-B: FIG. 33A shows purification of gH/gL/gp42_NP_C14 (SEQ IDNO: 230) using the Superose 6 size exclusion chromatography. The arrowdepicts the fractions collected from the peak with a denaturingcoomassie gel analysis and a western blot analysis using anti-ferritinantibodies. FIG. 33B is a dynamic light scattering analysis of thesample in FIG. 33A, which shows the particle size radius of 16.9 nm.

FIG. 34 : An SDS reducing coomassie gel on the left shows the purifiedsingle-chain gH/gL/gp42-His product (SEQ ID NO: 226). The protein waspurified using Nickel affinity chromatography. On the right is a 2.9Angstrom crystal structure of the single-chain gH/gL/gp42-His product(SEQ ID NO: 226). Gp42 (in dark gray and indicated with arrows)interacts with the gH/gL heterodimer.

FIG. 35A-E: A cartoon of a single-chain construct of gH/gL/gp42 fused toferritin (as in each of SEQ ID NOs: 227-231) is shown in FIG. 35A. Thefusion between each protein is via a flexible amino acid linker or arigid amino acid linker specified above. The single-chain gH/gL/gp42molecule will assure a 1:1:1 ratio of heterotrimer formation on thenanoparticle. The crystal structure of this heterotrimer has been solvedto show that the single-chain gH/gL/gp42 can adopt a heterotrimerformation similar to wild-type gH, gL, and gp42 proteins found in nature(FIG. 35B; see also FIG. 34 ). FIG. 35C is a model of how thissingle-chain gH/gL/gp42 heterotrimer is displayed on the nanoparticlethrough the fusion with ferritin. There are twenty-four copies of thesingle-chain gH/gL/gp42 displayed on a single nanoparticle. FIG. 35Dshows the purification after expression of SEQ ID NOs: 227 in 293Expicells. A denaturing SDS Coomassie gel shows the gH/gL/gp42 fused toferritin to be above 150 kD with glycosylation. FIG. 35E shows negativestain electron microscopy analysis of the purified product, indicatingthat the single-chain gH/gL/gp42 fused to ferritin can successfully formnanoparticles displaying the gH/gL/gp42 antigens on the surface.

DETAILED DESCRIPTION

EBV polypeptides are provided, which can be antigenic when administeredalone, with adjuvant as a separate molecule, and/or as part of ananoparticle (e.g., ferritin particle or lumazine synthase particle),which can be self-adjuvanting. Such polypeptides and compositionscomprising such polypeptides can be used to elicit antibody responsesagainst Epstein Barr virus (EBV). The EBV polypeptide can comprise a gL,gH, gL/gH, gp220, or gp42 polypeptide, or combinations thereof, and amultimerization domain such as a ferritin. The ferritin may comprise amutation replacing a surface-exposed amino acid with a cysteine, whichcan facilitate conjugating immune-stimulatory moieties to the ferritinvia the cysteine. Such conjugation may eliminate or reduce the need forseparately administered adjuvant, and may also potentially reduce theamount of adjuvant/immune-stimulatory moiety needed to elicit an immuneresponse to the EBV polypeptide. In some embodiments, an antigenic EBVpolypeptide comprising (i) an EBV polypeptide, and (ii-a) a ferritincomprising a surface-exposed cysteine, or (ii-b) a ferritin and an N- orC-terminal linker comprising a cysteine is provided. Any of the EBVpolypeptides described herein can be combined with any of the ferritinsdescribed below. Nucleic acids that encode the polypeptides describedherein are also provided.

A. Definitions

As used herein, an “EBV polypeptide” refers to a polypeptide comprisingall or part of an amino acid sequence encoded by EBV. Similarly, gL, gH,gp42, and gp220 polypeptides refer to polypeptides comprising all orpart of a gL, gH, gp42, or gp220 amino acid sequence, respectively,encoded by EBV. Polypeptides with, e.g., at least 80% identity to anEBV-encoded polypeptide will necessarily comprise part of theEBV-encoded polypeptide. The terms “gL polypeptide,” “gH polypeptide,”“gp42 polypeptide,” and “gp220 polypeptide” are used interchangeablywith “EBV gL polypeptide,” “EBV gH polypeptide,” “EBV gp42 polypeptide,”and “EBV gp220 polypeptide,” respectively. Immunization with an EBVpolypeptide as part or all of an antigenic polypeptide may conferprotection from infection with EBV. Unless the context dictatesotherwise, any polypeptide disclosed herein comprising an EBVpolypeptide can comprise all or part of multiple sequences encoded byEBV (for example, all or part of gL and gH of EBV, or all or part of gL,gH, and gp42 of EBV).

As used herein, a “monomer,” or “monomer construct” refers to aconstruct expressed as a single-chain protein. A monomer may comprise gLand gH of EBV expressed in a single chain, or gL, gH, and gp42 of EBVexpressed in a single chain.

As used herein, a “trimer,” or “trimer construct” refers to a constructcomprising gL and/or gH of EBV together with a trimerization domain,such as a foldon trimerization domain derived from T4 phage fibritin.Other trimerization domains, such as the human collagen XVIIItrimerization domain (see, e.g., Alvarez-Cienfuegos et al., ScientificReports 2016; 6:28643) and the L1ORF1p trimerization domain (see, e.g.,Khazina et al., Proc Natl Acad Sci USA 2009 Jan. 12; 106(3):731-36) arealso known in the art and can be used in trimeric constructs.

“Ferritin” or “ferritin protein,” as used herein, refers to a proteinwith detectable sequence identity to H. pylori ferritin (SEQ ID NO: 208or 209) or another ferritin discussed herein, such as P. furiosusferritin, Trichoplusia ni ferritin, or human ferritin, that serves tostore iron, e.g., intracellularly or in tissues or to carry iron in thebloodstream. Such exemplary ferritins, including those that occur as twopolypeptide chains, known as the heavy and light chains (e.g., T. ni andhuman ferritin), are discussed in detail below. In some embodiments, aferritin comprises a sequence with at least 15%, 20%, 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% identity to aferritin sequence disclosed herein, e.g., in Table 2 (Sequence Table). Aferritin may be a fragment of a full-length naturally-occurringsequence.

“Wild-type ferritin,” as used herein, refers to a ferritin whosesequence consists of a naturally-occurring sequence. Ferritins alsoinclude full-length ferritin or a fragment of ferritin with one or moredifferences in its amino acid sequence from a wild-type ferritin.

As used herein, a “ferritin monomer” refers to a single ferritinmolecule (or, where applicable, a single ferritin heavy or light chain)that has not assembled with other ferritin molecules. A “ferritinmultimer” comprises multiple associated ferritin monomers. A “ferritinprotein” includes monomeric ferritin and multimeric ferritin.

As used herein, “ferritin particle,” refers to ferritin that hasself-assembled into a globular form. Ferritin particles are sometimesreferred to as “ferritin nanoparticles” or simply “nanoparticles”. Insome embodiments, a ferritin particle comprises 24 ferritin monomers(or, where applicable, 24 total heavy and light chains).

“Hybrid ferritin,” as used herein, refers to ferritin comprising H.pylori ferritin with an amino terminal extension of bullfrog ferritin.An exemplary sequence used as an amino terminal extension of bullfrogferritin appears as SEQ ID NO: 217. In hybrid ferritin, the aminoterminal extension of bullfrog ferritin can be fused to H. pyloriferritin such that immune-stimulatory moiety attachment sites aredistributed evenly on the ferritin particle surface. “Bullfrog linker”as used herein is a linker comprising the sequence of SEQ ID NO: 217.Hybrid ferritin is also sometimes referred to as “bfpFerr” or “bfpferritin.” Any of the constructs comprising a bullfrog sequence can beprovided without the bullfrog sequence, such as, for example, without alinker or with an alternative linker. Exemplary bullfrog linkersequences are provided in Table 2. Where Table 2 shows a bullfroglinker, the same construct may be made without a linker or with analternative linker.

“N-glycan,” as used herein, refers to a saccharide chain attached to aprotein at the amide nitrogen of an N (asparagine) residue of theprotein. As such, an N-glycan is formed by the process ofN-glycosylation. This glycan may be a polysaccharide.

“Glycosylation,” as used herein, refers to the addition of a saccharideunit to a protein.

“Immune response,” as used herein, refers to a response of a cell of theimmune system, such as a B cell, T cell, dendritic cell, macrophage orpolymorphonucleocyte, to a stimulus such as an antigen or vaccine. Animmune response can include any cell of the body involved in a hostdefense response, including for example, an epithelial cell thatsecretes an interferon or a cytokine. An immune response includes, butis not limited to, an innate and/or adaptive immune response. As usedherein, a “protective immune response” refers to an immune response thatprotects a subject from infection (e.g., prevents infection or preventsthe development of disease associated with infection). Methods ofmeasuring immune responses are well known in the art and include, forexample, by measuring proliferation and/or activity of lymphocytes (suchas B or T cells), secretion of cytokines or chemokines, inflammation,antibody production and the like. An “antibody response” is an immuneresponse in which antibodies are produced.

As used herein, an “antigen” refers to an agent that elicits an immuneresponse, and/or an agent that is bound by a T cell receptor (e.g., whenpresented by an WIC molecule) or to an antibody (e.g., produced by a Bcell) when exposed or administered to an organism. In some embodiments,an antigen elicits a humoral response (e.g., including production ofantigen-specific antibodies) in an organism. Alternatively, oradditionally, in some embodiments, an antigen elicits a cellularresponse (e.g., involving T-cells whose receptors specifically interactwith the antigen) in an organism. A particular antigen may elicit animmune response in one or several members of a target organism (e.g.,mice, rabbits, primates, humans), but not in all members of the targetorganism species. In some embodiments, an antigen elicits an immuneresponse in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ofthe members of a target organism species. In some embodiments, anantigen binds to an antibody and/or T cell receptor, and may or may notinduce a particular physiological response in an organism. In someembodiments, for example, an antigen may bind to an antibody and/or to aT cell receptor in vitro, whether or not such an interaction occurs invivo. In some embodiments, an antigen reacts with the products ofspecific humoral or cellular immunity, including those induced byheterologous immunogens. Antigens include antigenic ferritin proteinscomprising ferritin (e.g., comprising one or more mutations) and anon-ferritin polypeptide as described herein.

An “immune-stimulatory moiety,” as used herein, refers to a moiety thatis covalently attached to a ferritin or antigenic ferritin polypeptideand that can activate a component of the immune system (either alone orwhen attached to ferritin or antigenic ferritin polypeptide). Exemplaryimmune-stimulatory moieties include agonists of toll-like receptors(TLRs), e.g., TLR 4, 7, 8, or 9. In some embodiments, animmune-stimulatory moiety is an adjuvant.

“Adjuvant,” as used herein, refers to a substance or vehicle thatnon-specifically enhances the immune response to an antigen. Adjuvantscan include, without limitation, a suspension of minerals (e.g., alum,aluminum hydroxide, or phosphate) on which antigen is adsorbed; awater-in-oil or oil-in-water emulsion in which antigen solution isemulsified in mineral oil or in water (e.g., Freund's incompleteadjuvant). Sometimes killed mycobacteria is included (e.g., Freund'scomplete adjuvant) to further enhance antigenicity. Immuno-stimulatoryoligonucleotides (e.g., a CpG motif) can also be used as adjuvants (forexample, see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants can alsoinclude biological molecules, such as Toll-Like Receptor (TLR) agonistsand costimulatory molecules. An adjuvant may be administered as aseparate molecule in a composition or covalently bound (conjugated) toferritin or an antigenic ferritin polypeptide.

An “antigenic EBV polypeptide” is used herein to refer to a polypeptidecomprising all or part of an EBV amino acid sequence of sufficientlength that the molecule is antigenic with respect to EBV. Antigenicitymay be a feature of the EBV sequence as part of a construct furthercomprising a heterologous sequence, such as a ferritin or lumazinesynthase protein and/or immune-stimulatory moiety. That is, if an EBVsequence is part of a construct further comprising a heterologoussequence, then it is sufficient that the construct can serve as anantigen that generates anti-EBV antibodies, regardless of whether theEBV sequence without the heterologous sequence could do so.

“Antigenic ferritin polypeptide” and “antigenic ferritin protein” areused interchangeably herein to refer to a polypeptide comprising aferritin and a non-ferritin polypeptide (e.g., an EBV polypeptide) ofsufficient length that the molecule is antigenic with respect to thenon-ferritin polypeptide. The antigenic ferritin polypeptide may furthercomprise an immune-stimulatory moiety. Antigenicity may be a feature ofthe non-ferritin sequence as part of the larger construct. That is, itis sufficient that the construct can serve as an antigen against thenon-ferritin polypeptide, regardless of whether the non-ferritinpolypeptide without the ferritin (and immune-stimulatory moiety ifapplicable) could do so. In some embodiments, the non-ferritinpolypeptide is an EBV polypeptide, in which case the antigenic ferritinpolypeptide is also an “antigenic EBV polypeptide.” To be clear,however, an antigenic EBV polypeptide does not need to compriseferritin. “Antigenic polypeptide” is used herein to refer to apolypeptide which is either or both of an antigenic ferritin polypeptideand an antigenic EBV polypeptide.

“Self-adjuvanting,” as used herein, refers to a composition orpolypeptide comprising a ferritin and an immune-stimulatory moietydirectly conjugated to the ferritin so that the ferritin andimmune-stimulatory moiety are in the same molecular entity. An antigenicferritin polypeptide comprising a non-ferritin polypeptide may beconjugated to an immune-stimulatory moiety to generate aself-adjuvanting polypeptide.

A “surface-exposed” amino acid, as used herein, refers to an amino acidresidue in a protein (e.g., a ferritin) with a side chain that can becontacted by solvent molecules when the protein is in its nativethree-dimensional conformation after multimerization, if applicable.Thus, for example, in the case of ferritin that forms a 24-mer, asurface-exposed amino acid residue is one whose side chain can becontacted by solvent when the ferritin is assembled as a 24-mer, e.g.,as a ferritin multimer or ferritin particle.

As used herein, a “subject” refers to any member of the animal kingdom.In some embodiments, “subject” refers to humans. In some embodiments,“subject” refers to non-human animals. In some embodiments, subjectsinclude, but are not limited to, mammals, birds, reptiles, amphibians,fish, insects, and/or worms. In certain embodiments, the non-humansubject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey,a dog, a cat, a sheep, cattle, a primate, and/or a pig). In someembodiments, a subject may be a transgenic animal,genetically-engineered animal, and/or a clone. In certain embodiments ofthe present invention the subject is an adult, an adolescent or aninfant. In some embodiments, terms “individual” or “patient” are usedand are intended to be interchangeable with “subject”.

As used herein, the term “vaccination” or “vaccinate” refers to theadministration of a composition intended to generate an immune response,for example to a disease-causing agent. Vaccination can be administeredbefore, during, and/or after exposure to a disease-causing agent, and/orto the development of one or more symptoms, and in some embodiments,before, during, and/or shortly after exposure to the agent. In someembodiments, vaccination includes multiple administrations,appropriately spaced in time, of a vaccinating composition.

The disclosure describes nucleic acid sequences and amino acid sequenceshaving a certain degree of identity to a given nucleic acid sequence oramino acid sequence, respectively (a references sequence).

“Sequence identity” between two nucleic acid sequences indicates thepercentage of nucleotides that are identical between the sequences.“Sequence identity” between two amino acid sequences indicates thepercentage of amino acids that are identical between the sequences.

The terms “% identical”, “% identity” or similar terms are intended torefer, in particular, to the percentage of nucleotides or amino acidswhich are identical in an optimal alignment between the sequences to becompared. Said percentage is purely statistical, and the differencesbetween the two sequences may be but are not necessarily randomlydistributed over the entire length of the sequences to be compared.Comparisons of two sequences are usually carried out by comparing saidsequences, after optimal alignment, with respect to a segment or “windowof comparison”, in order to identify local regions of correspondingsequences. The optimal alignment for a comparison may be carried outmanually or with the aid of the local homology algorithm by Smith andWaterman, 1981, Ads App. Math. 2, 482, with the aid of the localhomology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443,with the aid of the similarity search algorithm by Pearson and Lipman,1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computerprograms using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST Nand TFASTA in Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Drive, Madison, Wis.).

Percentage identity is obtained by determining the number of identicalpositions at which the sequences to be compared correspond, dividingthis number by the number of positions compared (e.g., the number ofpositions in the reference sequence) and multiplying this result by 100.

In some embodiments, the degree of identity is given for a region whichis at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90% or about 100% of the entire length of thereference sequence. For example, if the reference nucleic acid sequenceconsists of 200 nucleotides, the degree of identity is given for atleast about 100, at least about 120, at least about 140, at least about160, at least about 180, or about 200 nucleotides, in some embodimentsin continuous nucleotides. In some embodiments, the degree of identityis given for the entire length of the reference sequence.

Nucleic acid sequences or amino acid sequences having a particulardegree of identity to a given nucleic acid sequence or amino acidsequence, respectively, may have at least one functional property ofsaid given sequence, e.g., and in some instances, are functionallyequivalent to said given sequence. One important property includes theability to act as a cytokine, in particular when administered to asubject. In some embodiments, a nucleic acid sequence or amino acidsequence having a particular degree of identity to a given nucleic acidsequence or amino acid sequence is functionally equivalent to said givensequence.

As used herein, the term “kit” refers to a packaged set of relatedcomponents, such as one or more compounds or compositions and one ormore related materials such as solvents, solutions, buffers,instructions, or desiccants.

B. Antigenic EBV Polypeptides Comprising gL and gH Polypeptides

EBV has three glycoproteins, glycoprotein B (gB), gH, and gL, that formthe core membrane fusion machinery to allow viral penetration into acell. gL and gH have been previously described, for example, in Matsuuraet al., Proc Natl Acad Sci USA. 2010 Dec. 28; 107(52):22641-6. Monomersand trimers of gL and gH for use as vaccines have been described, forexample, in Cui et al., Vaccine. 2016 Jul. 25; 34(34):4050-5. The gH andgL proteins associate to form a heterodimeric complex considerednecessary for efficient membrane fusion and binding to epithelial cellreceptors required for viral entry.

Disclosed herein are antigenic polypeptides comprising EBV gL and EBVgH. In some embodiments, the polypeptide exists as a single-chain. Insome embodiments, the polypeptide forms a trimer, e.g., throughtrimerization of a trimerization domain, such as a T4 phage fibritintrimerization domain. In some embodiments, the polypeptide forms ananoparticle (e.g., ferritin or lumazine synthase particle), e.g.,through multimerization of a ferritin or lumazine synthase. In someembodiments, an antigenic EBV polypeptide according to this disclosurecomprises an EBV gL polypeptide and an EBV gH polypeptide, and a linkerhaving a length of at least 15 amino acids separating the EBV gLpolypeptide and the EBV gH polypeptide. It has been found that arelatively long linker can provide benefits such as improved expressionand/or immunogenicity.

In some embodiments, the EBV gH and/or gL polypeptides are full-lengthgH and/or gL (for exemplary full-length sequences, see GenBank AccessionNos. CEQ35765.1 and YP_001129472.1, respectively). In some embodiments,the EBV gH and/or gL polypeptides are fragments of gH and/or gL. In someembodiments, the gL polypeptide is a gL(D7) construct with a 7-aminoacid deletion at the end of the gL C terminus. In some embodiments, thegH polypeptide comprises a mutation at C137, such as a C137A mutation.In some embodiments, the C137 mutation removes a native, unpairedcysteine to avoid non-specific conjugation. In some embodiments, the gHpolypeptide comprises a mutation to remove a cysteine corresponding tocysteine 137 of SEQ ID NO: 37, such as a C137A mutation. In someembodiments, the C137 mutation removes a native, unpaired cysteine toavoid non-specific conjugation.

In some embodiments, the EBV gL polypeptide comprises an amino acidsequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 36. In some embodiments, the EBV gH polypeptidecomprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%,98%, 99%, or 100% identity to SEQ ID NO: 37.

In some embodiments, a mammalian leader sequence (also known as a signalsequence) is appended N-terminally to an EBV polypeptide such as a gH orgL polypeptide, e.g., at the N-terminus of the polypeptide. In someembodiments, a mammalian leader sequence results in secretion of aprotein when expressed in mammalian cells.

Native EBV gH and/or gL sequences are shown in GenBank Accession No.NC_009334.1 (Human herpesvirus 4, complete genome, dated 26 Mar. 2010).For some of the constructs disclosed herein, amino acids 23-137 of thegL amino acid sequence in NC_009334.1 was used as the gL polypeptide,and the native signal peptide (amino acids 1-22 of the NCBI sequence)was replaced with an IgG K leader sequence. For some of the constructs,amino acids 19-678 of the gH amino acid sequence in NC_009334.1 was usedas the gH polypeptide. In some embodiments, the gL and gH were linkedvia a linker as shown in the table of sequences herein.

In some embodiments, gL and gH polypeptides are expressed as asingle-chain monomer. In some embodiments, the monomer compositioncomprises or consists of a sequence shown in the Sequence Table anddenoted in the description as “monomer”. A single-chain comprising gLand gH polypeptides may be referred to as “gL/gH,” which can be usedinterchangeably with “gH_gL,” “gL_gH,” or “gL/gH.”

In some embodiments, gL and gH are provided as a trimer. In someembodiments, a trimerization domain is placed after (C-terminal to) thegH sequence and in some embodiments, this is followed by a His6 (SEQ IDNO: 243) sequence. The foldon trimerization domain is exemplary, as anytrimerization domain known in the art can be used, such as collagen orL1ORF1p trimerization domains referenced herein. A gL and gH trimer hasbeen shown to induce higher serum neutralization titers relative to a gLand gH monomer using peripheral blood human naïve B cells (see, forexample, Cui et al., Vaccine. 2016 Jul. 25; 34(34):4050-5).

In some embodiments, a gL/gH trimer has an amino acid sequencecomprising or consisting of a sequence shown in the Sequence Table anddenoted in the description as “trimer.”

The gL/gH polypeptide can be combined with any of the ferritins orlumazine synthases discussed herein. For example, in some embodiments,an antigenic EBV polypeptide comprises a monomer or trimer gL/gHpolypeptide (+/−gp42 and/or gp220) and i) a heavy or light chainferritin (e.g., T. ni heavy or light chain ferritin); or ii) a ferritin,optionally comprising a surface-exposed cysteine.

Additionally, in some embodiments, any antigenic EBV polypeptidecomprising an EBV gL/gH polypeptide and a ferritin can be present in acomposition comprising another polypeptide disclosed herein.

C. Antigenic EBV Polypeptides Comprising a Gp220 Polypeptide

In some embodiments, an antigenic EBV polypeptide comprises a gp220polypeptide. A gp220-hybrid bullfrog/H. pylori ferritin nanoparticle hasbeen previously described in Kanekiyo Cell. 2015 Aug. 27;162(5):1090-100. This nanoparticle did not comprise a mutation providinga surface-exposed cysteine or a linker comprising a cysteine, amongother differences from certain ferritins described herein.

In some embodiments, the gp220 polypeptide comprises an amino acidsequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 38.

In some embodiments, a mammalian leader sequence (also known as a signalsequence) is N-terminally appended to a gp220 polypeptide. In someembodiments, a mammalian leader sequence results in secretion of aprotein when expressed in mammalian cells.

The gp220 polypeptide can be combined with any of the ferritins orlumazine synthases discussed herein. For example, in some embodiments,an antigenic EBV polypeptide comprises a gp220 polypeptide (+/−gL/gHand/or gp42) and i) a heavy or light chain ferritin (e.g., T. ni heavyor light chain ferritin); or ii) a ferritin, optionally comprising asurface-exposed cysteine as described herein.

Additionally, in some embodiments, any antigenic EBV polypeptidecomprising a gp220 polypeptide and a ferritin can be present in acomposition comprising another polypeptide disclosed herein.

D. Antigenic EBV Polypeptides Comprising a Gp42 Polypeptide

In some embodiments, an antigenic EBV polypeptide comprises a gp42polypeptide. An exemplary gp42 sequence is provided as SEQ ID NO: 34. Afurther exemplary gp42 sequence, suitable for inclusion in fusions e.g.with gL and gH polypeptides, is provided as SEQ ID NO: 239. Anotherexemplary gp42 sequence, suitable for inclusion in fusions e.g. with gLand gH polypeptides, is provided as SEQ ID NO: 240.

In some embodiments, the gp42 polypeptide comprises an amino acidsequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 34. In some embodiments, the gp42 polypeptidecomprises an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%,98%, 99%, or 100% identity to SEQ ID NO: 239. In some embodiments, thegp42 polypeptide comprises an amino acid sequence with at least 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 240.

In some embodiments, a mammalian leader sequence (also known as a signalsequence) is N-terminally appended to a gp42 polypeptide. In someembodiments, a mammalian leader sequence results in secretion of aprotein when expressed in mammalian cells. An exemplary leader sequenceis amino acids 1-22 of SEQ ID NO: 226.

In some embodiments, an antigenic EBV polypeptide comprising a gH and/orgL polypeptide further comprises a gp42 polypeptide. Any of the EBVpolypeptides comprising a gH and/or gL polypeptide described above canfurther comprise a gp42 polypeptide. In some embodiments, the gp42polypeptide is located C-terminal to the gH and/or gL polypeptide(s), asexemplified in SEQ ID NOs: 21 and 226-231. In some embodiments, the gp42polypeptide is located N-terminal to a ferritin, also as exemplified inSEQ ID NOs: 21 and 227-231. Thus, for example, an antigenic EBVpolypeptide may comprise, in N- to C-terminal orientation, a gLpolypeptide, a gH polypeptide, a gp42 polypeptide, and optionally aferritin. Linkers such as those described herein can separate the gp42polypeptide from EBV polypeptides and/or ferritins located N-terminaland/or C-terminal thereto. In some embodiments, a linker separates eachEBV polypeptide in an antigenic ferritin polypeptide (e.g., a gLpolypeptide, a gH polypeptide, and a gp42 polypeptide), and a furtherlinker may be present between the ferritin if present and the EBVpolypeptide proximal thereto (e.g., a gp42 polypeptide).

In some embodiments, a linker having a length of at least 15 amino acidsseparates the EBV gH polypeptide and the EBV gp42 polypeptide. Such alinker may have a length of 15 to 60 amino acids, 20 to 60 amino acids,30 to 60 amino acids, 40 to 60 amino acids, 30 to 50 amino acids, or 40to 50 amino acids. In some embodiments, the linker comprises an aminoacid sequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 234.

In some embodiments, where gp42 and ferritin are present in apolypeptide, a linker separates the EBV gp42 polypeptide and theferritin. Such a linker may have a length of at least 15 amino acids orhas a length of 15 to 60 amino acids, 20 to 60 amino acids, 30 to 60amino acids, 40 to 60 amino acids, 30 to 50 amino acids, or 40 to 50amino acids. In some embodiments, such a linker comprises an amino acidsequence with at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to any one of SEQ ID NOs: 233, 234, 235, 236, 237, or 238.

The gp42 polypeptide can be combined with any of the ferritins orlumazine synthases discussed herein. For example, in some embodiments, apolypeptide comprises a gp42 polypeptide (+/−gL/gH and/or gp220) and aheavy or light chain ferritin (e.g., T. ni heavy or light chainferritin); or ii) ferritin, optionally comprising a surface-exposedcysteine as described herein.

In some embodiments, the antigenic EBV polypeptide comprises a sequencewith at least 80%, 85%, 90%, 95%, 98%, or 99% identity to amino acids23-1078 of SEQ ID NO: 226. In some embodiments, the antigenic EBVpolypeptide comprises a sequence with 80%, 85%, 90%, 95%, 98%, or 99%identity to amino acids 1-1078 of SEQ ID NO: 226. In some embodiments,the antigenic EBV polypeptide comprises a sequence with at least 80%,85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID NOs: 226, 227,228, 229, 230, or 231, optionally lacking the leader sequence (e.g.,lacking any or all of amino acids 1-22 of these sequences).

Additionally, in some embodiments, any antigenic EBV polypeptidecomprising a gp42 polypeptide and a ferritin can be present in acomposition comprising another polypeptide disclosed herein.

E. Linkers

In some embodiments, an antigenic EBV polypeptide comprises a linkerbetween gL and gH polypeptides. In some embodiments, an antigenic EBVpolypeptide comprises a linker between an EBV polypeptide and a ferritinor lumazine synthase. The following features are described with respectto either of such linkers, although the present invention provides thata relatively long linker between the gL and gH sequences may provide anincrease in immunogenicity. Any linker may be used; for example, in someembodiments, the linker is 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids inlength. In some embodiments, the linker is about 2-4, 2-6, 2-8, 2-10,2-12, or 2-14 amino acids in length. In some embodiments, the linker isa peptide linker, which can facilitate expression of the antigenicferritin polypeptide as a fusion protein (e.g., from a single openreading frame). In some embodiments, the linker is a glycine-serinelinker. In some embodiments, the glycine-serine linker is GS, GGGS (SEQID NO: 244), 2XGGGS (i.e., GGGSGGGS) (SEQ ID NO: 245), or 5XGGGS (SEQ IDNO: 246). In some embodiments, the linker between the EBV polypeptideand ferritin is GS, GGGS (SEQ ID NO: 244), 2XGGGS (i.e., GGGSGGGS) (SEQID NO: 245), or 5XGGGS (SEQ ID NO: 246).

In some embodiments, the linker is at least 15 amino acids in length. Insome embodiments, the linker is at least 25 amino acids in length. Insome embodiments, the linker is at least 30 amino acids in length. Insome embodiments, the linker is at least 35 amino acids in length. Insome embodiments, the linker is at least 40 amino acids in length. Insome embodiments, the linker is less than or equal to 60 amino acids inlength. In some embodiments, the linker is less than or equal to 50amino acids in length. In some embodiments, the linker is about 16, 28,40, 46, or 47 amino acids in length. In some embodiments, the linker isflexible. In some embodiments, the linker comprises a cysteine, e.g.,for use as a site for conjugation of an immune-stimulatory moiety (e.g.,adjuvant); an exemplary linker comprising a cysteine is provided as SEQID NO: 225. In some embodiments, the linker comprises a sequence with atleast 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 225, and furthercomprises a cysteine corresponding to the cysteine in SEQ ID NO: 225. Insome embodiments, the linker comprises at least 25 amino acids (e.g., 25to 60 amino acids), wherein a cysteine is located at a position rangingfrom the 8^(th) amino acid from the N-terminus to the 8^(th) amino acidfrom the C-terminus, or within 10 amino acids of the central residue orbond of the linker.

In some embodiments, the linker comprises glycine (G) and/or serine (S)amino acids. In some embodiments, the linker comprises or consists ofglycine (G), serine (S), asparagine (N), and/or alanine (A) amino acids,and optionally a cysteine as discussed above. In some embodiments, thelinker comprises an amino acid sequence with at least 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 222. In someembodiments, the linker comprises GGGGSGGGGSGGGGSG (SEQ ID NO: 28),GGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 29),GGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGSGGSG (SEQ ID NO: 30), or GS.In some embodiments, the linker comprises FR1 (SEQ ID NO: 31) or FR2(SEQ ID NO: 32). In some embodiments, the linker comprises SEQ ID NO:233-238.

In some embodiments, a linker comprising a cysteine as a conjugationsite for an immune-stimulatory moiety such as an adjuvant is used in aconstruct comprising a ferritin molecule lacking an unpaired,surface-exposed cysteine, or in a construct comprising a ferritinmolecule comprising an unpaired, surface-exposed cysteine.

In some embodiments, the linker is a cysteine-thrombin-histidine linker.In some embodiments, this linker is used to directly conjugate an EBVpolypeptide to ferritin via click chemistry. An exemplary sequencecomprising a cysteine-thrombin-histidine linker is SEQ ID NO: 39. Clickchemistry suitable for conjugation reactions involving thecysteine-thrombin-histidine linker is discussed herein.

In some embodiments, a construct does not comprise a linker. In someembodiments, a construct comprises one linker. In some embodiments, aconstruct comprises two or more than two linkers.

In some embodiments, the construct comprises a linker between gH and gLbut not between the polypeptide and ferritin or vice versa. In someembodiments, the construct only comprises a linker between thepolypeptide and ferritin.

F. Antigenic EBV Polypeptides Comprising an EBV Polypeptide and Ferritinor Lumazine Synthase

In some embodiments, an antigenic EBV polypeptide is provided,comprising an EBV polypeptide and ferritin. The EBV polypeptide can beany of the EBV polypeptides described herein, such as a gL, gH, gL/gH,gp220, gp42 polypeptide, or combinations thereof. The ferritin componentof the polypeptide may be a ferritin from any species, and may or maynot have mutations, such as a mutation replacing a surface-exposed aminoacid with a cysteine as described herein. In some embodiments, thepolypeptide comprises the amino acids of any one of SEQ ID NOS: 1-27.

In some embodiments, the ferritin in the polypeptide is a wild-typeferritin. In some embodiments, the ferritin is bacterial, insect,fungal, bird, or mammalian. In some embodiments, the ferritin is human.In some embodiments, the ferritin is bacterial.

In some embodiments, the ferritin is a light chain and/or heavy chainferritin. In some embodiments, the ferritin is an insect ferritin, suchas Trichoplusia ni heavy chain ferritin (SEQ ID NO: 211) or Trichoplusiani light chain ferritin (SEQ ID NO: 212). In some embodiments, theferritin is a human ferritin, such as human heavy chain ferritin (SEQ IDNO: 214 or FTH1, GENE ID No: 2495) or human light chain ferritin (SEQ IDNO: 215 or FTL, GENE ID No: 2512). In some embodiments, a ferritinnanoparticle comprises 24 total subunits of heavy chain ferritin andlight chain ferritin, such as in human or Trichoplusia ni ferritinnanoparticles. T. ni ferritin nanoparticles can comprise 12 subunits ofheavy chain ferritin and 12 subunits of light chain ferritin.

In some embodiments, an antigenic EBV polypeptide comprises a lightchain ferritin and an EBV polypeptide. In some embodiments, an antigenicEBV polypeptide comprises a heavy chain ferritin and an EBV polypeptide.In some embodiments, an antigenic EBV polypeptide comprising a lightchain ferritin and an EBV polypeptide can assemble with a heavy chainferritin that is not linked to an EBV polypeptide. In some embodiments,an antigenic EBV polypeptide comprising a heavy chain ferritin and anEBV polypeptide can assemble with a light chain ferritin that is notlinked to an EBV polypeptide. A ferritin not linked to an EBVpolypeptide (or, more generally, a non-ferritin polypeptide) may bereferred as a “naked ferritin.”

In some embodiments, an antigenic polypeptide comprising a heavy chainferritin and a polypeptide can assemble with an antigenic polypeptidecomprising a light chain ferritin and an EBV polypeptide to allowpresentation of two of the same or different non-ferritin polypeptideson a single ferritin nanoparticle. In some embodiments, the twodifferent non-ferritin polypeptides are EBV polypeptides. In someembodiments, the two different non-ferritin polypeptides are encoded byEBV and a different infectious agent. In some embodiments, the differentnon-ferritin polypeptide from a different infectious agent is from avirus or bacterium.

In some embodiments, an antigenic polypeptide comprising a heavy chainferritin and a non-ferritin polypeptide can assemble with a polypeptidecomprising a light chain ferritin and a non-ferritin polypeptide toproduce a bivalent composition.

In some embodiments, an antigenic polypeptide comprises a light chainferritin and a gp220 and/or gp42 polypeptide. In some embodiments, anantigenic polypeptide comprises a heavy chain ferritin and a gp220and/or gp42 polypeptide.

In some embodiments, an antigenic polypeptide comprises a light chainferritin and a single-chain gL and gH polypeptide. In some embodiments,an antigenic polypeptide comprises a heavy chain ferritin and asingle-chain gL and gH polypeptide.

In some embodiments, an antigenic polypeptide comprising a light chainferritin and a gp220 and/or gp42 polypeptide assembles with an antigenicpolypeptide comprising a heavy chain ferritin and a single-chain gL andgH polypeptide.

In some embodiments, an antigenic polypeptide comprising a heavy chainferritin and a gp220 and/or gp42 polypeptide assembles with an antigenicpolypeptide comprising a light chain ferritin and a single-chain gL andgH polypeptide. In some embodiments, twelve (12) gp220 and/or gp42polypeptides and twelve (12) single-chain gL and gH polypeptides arecomprised in an assembled ferritin nanoparticle, as in the case of anassembled T. ni ferritin nanoparticle.

Any type of ferritin nanoparticle(s) that comprises both gp220 and/orgp42 and single-chain gL and gH polypeptides may be referred to as a“bivalent” or “bivalent EBV” particle or construct. A compositioncomprising a gL and gH trimer together with a ferritin that comprisesgp220 and/or gp42 would also be a bivalent EBV composition.

In some embodiments, the ferritin is H. pylori ferritin (see SEQ ID NO:208 or 209 for an exemplary H. pylori ferritin sequence), optionallywith one or more mutations such as those described herein. In someembodiments, the lower sequence homology between H. pylori ferritin (orother bacterial ferritins) and human ferritin may decrease the potentialfor autoimmunity when used as a vaccine platform (see Kanekiyo et al.,Cell 162, 1090-1100 (2015)).

In some embodiments, a nanoparticle is provided comprising an antigenicEBV polypeptide as disclosed herein comprising an EBV polypeptide and aferritin.

1. Ferritin Mutations

In some embodiments, the ferritin comprises one or more mutations aredisclosed herein. In some embodiments, the one or more mutationscomprise changes to the amino acid sequence of a wild-type ferritinand/or an insertion, e.g., at the N- or C-terminus. In some embodiments,one, two, three, four, five, or more different amino acids are mutatedin the ferritin as compared to wild-type ferritin (in some embodiments,in addition to any N-terminal insertion). The one or more mutations canchange functional properties of the ferritin, e.g., as discussed indetail below. In general, a mutation simply refers to a difference inthe sequence (such as a substituted, added, or deleted amino acidresidue or residues) relative to the corresponding wild-type ferritin.

2. Cysteine for Conjugation

In some embodiments, ferritin is mutated to provide a chemical handlefor conjugation of an immune-stimulatory moiety and/or EBV polypeptide.This can be achieved with a mutation replacing a surface-exposednon-cysteine amino acid with a cysteine. For the avoidance of doubt,language such as “replacing a surface-exposed amino acid with acysteine” necessarily implies that the surface-exposed amino acid in thewild-type or pre-mutation sequence is not cysteine. Another approach forproviding a chemical handle for conjugation of an immune-stimulatorymoiety or EBV polypeptide is to include a segment of amino acids, suchas a linker, N- or C-terminal to the ferritin, wherein the segment ofamino acids comprises a cysteine. In some embodiments, this cysteine(whether replacing a surface-exposed amino acid or in an N- orC-terminal linker) is unpaired, which means that it does not have anappropriate partner cysteine to form a disulfide bond. In someembodiments, this cysteine does not change the secondary structure offerritin. In some embodiments, this cysteine does not change thetertiary structure of ferritin.

In some embodiments, this cysteine can be used to conjugate agents, suchas immune-stimulatory moieties, to ferritin. In some embodiments, thiscysteine provides a free thiol group that is reactive. In someembodiments, agents conjugated to this cysteine on ferritin are exposedon the surface of an assembled ferritin particle. In some embodiments,this cysteine can interact with molecules and cells of the subject afteradministration while the ferritin particle is assembled.

In some embodiments, the presence of this cysteine allows conjugation ofone or more immune-stimulatory moieties, e.g., adjuvants. In someembodiments, conjugation of the immune-stimulatory moiety would notoccur in the absence of this cysteine.

In some embodiments, the non-cysteine amino acid that is replaced with acysteine is selected from E12, S72, A75, K79, S100, and S111 of H.pylori ferritin. Thus, in some embodiments, the surface-exposed aminoacid that is replaced in favor of cysteine is an amino acid residue thatcorresponds to E12, S26, S72, A75, K79, S100, or S111 of H. pyloriferritin. Analogous amino acids can be found in non-H. pylori ferritinby pair-wise or structural alignment. In some embodiments, thenon-cysteine amino acid that is replaced with a cysteine can be selectedfrom an amino acid that corresponds to S3, S19, S33, I82, A86, A102, andA120 of human light chain ferritin. In some embodiments, thesurface-exposed amino acid to be replaced with a cysteine is selectedbased on the understanding that if the native amino acid were replacedwith cysteine, it would be reactive in an assembled ferritin multimer orparticle and/or that this cysteine does not disrupt the stability of theferritin multimer or particle and/or that this cysteine does not lead toreduction in expression levels of ferritin.

In some embodiments, the ferritin comprises an E12C mutation. In someembodiments, the E12C residue can be used to conjugate agents (e.g.,immune-stimulatory moieties and/or EBV polypeptides) to ferritin. Insome embodiments, the E12C residue provides a free thiol group that isreactive. In some embodiments, agents conjugated to the E12C residue onferritin monomers are expressed on the surface on an assembled ferritinmultimer or particle. In some embodiments, twenty-four E12C residues(one from each monomer) are present on the surface of a ferritinmultimer or particle.

In some embodiments, the ferritin comprises an S26C mutation. In someembodiments, the S26C residue can be used to conjugate agents (e.g.,immune-stimulatory moieties and/or EBV polypeptides) to ferritin. Insome embodiments, the S26C residue provides a free thiol group that isreactive. In some embodiments, agents conjugated to the S26C residue onferritin monomers are expressed on the surface on an assembled ferritinmultimer or particle. In some embodiments, twenty-four S26C residues(one from each monomer) are present on the surface of a ferritinmultimer or particle.

In some embodiments, the ferritin comprises an S72C mutation. In someembodiments, the S72C residue can be used to conjugate agents (e.g.,immune-stimulatory moieties and/or EBV polypeptides) to ferritin. Insome embodiments, the S72C residue provides a free thiol group that isreactive. In some embodiments, agents conjugated to the S72C residue onferritin monomers are expressed on the surface on an assembled ferritinmultimer or particle. In some embodiments, twenty-four S72C residues(one from each monomer) are present on the surface of a ferritinmultimer or particle.

In some embodiments, the ferritin comprises an A75C mutation. In someembodiments, the A75C residue can be used to conjugate agents (e.g.,immune-stimulatory moieties and/or EBV polypeptides) to ferritin. Insome embodiments, the A75C residue provides a free thiol group that isreactive. In some embodiments, agents conjugated to the A75C residue onferritin monomers are expressed on the surface on an assembled ferritinmultimer or particle. In some embodiments, twenty-four A75C residues(one from each monomer) are present on the surface of a ferritinmultimer or particle.

In some embodiments, the ferritin comprises an K79C mutation. In someembodiments, the K79C residue can be used to conjugate agents (e.g.,immune-stimulatory moieties and/or EBV polypeptides) to ferritin. Insome embodiments, the K79C residue provides a free thiol group that isreactive. In some embodiments, agents conjugated to the K79C residue onferritin monomers are expressed on the surface on an assembled ferritinmultimer or particle. In some embodiments, twenty-four K79C residues(one from each monomer) are present on the surface of a ferritinmultimer or particle.

In some embodiments, the ferritin comprises an S100C mutation. In someembodiments, the S100C residue can be used to conjugate agents (e.g.,immune-stimulatory moieties and/or EBV polypeptides) to ferritin. Insome embodiments, the S100C residue provides a free thiol group that isreactive. In some embodiments, agents conjugated to the S100C residue onferritin monomers are expressed on the surface on an assembled ferritinmultimer or particle. In some embodiments, twenty-four S100C residues(one from each monomer) are present on the surface of a ferritinmultimer or particle.

In some embodiments, the ferritin comprises an S111C mutation. In someembodiments, the S111C residue can be used to conjugate agents (e.g.,immune-stimulatory moieties and/or EBV polypeptides) to ferritin. Insome embodiments, the S111C residue provides a free thiol group that isreactive. In some embodiments, agents conjugated to the S111C residue onferritin monomers are expressed on the surface on an assembled ferritinmultimer or particle. In some embodiments, twenty-four S111C residues(one from each monomer) are present on the surface of a ferritinmultimer or particle.

3. Removal of Internal Cysteine

In some embodiments, the ferritin comprises a mutation replacing aninternal cysteine with a non-cysteine amino acid. Removal of a nativeinternal cysteine residue can ensure that there is only one unpairedcysteine per ferritin monomer and avoid undesired reactions such asdisulfide formation and may result in a more stable and efficient result(e.g., adjuvant presentation). In some embodiments, C31 of H. pyloriferritin is replaced with a non-cysteine amino acid. In someembodiments, C31 of H. pylori ferritin is replaced with a serine (C31S),although any non-cysteine residue may be used, e.g., alanine, glycine,threonine, or asparagine. Analogous amino acids can be found in non-H.pylori ferritin by pair-wise or structural alignment. Thus, in someembodiments, the internal cysteine that is replaced in favor ofnon-cysteine is an amino acid residue that aligns with C31 of H. pyloriferritin. Exemplary ferritin sequences showing a C31S mutation are shownin SEQ ID NOS: 201-207. In some embodiments, when more than one internalcysteine is present in ferritin, two or more (e.g., each) internalcysteine is replaced with a non-cysteine amino acid, such as serine oran amino acid selected from serine, alanine, glycine, threonine, orasparagine.

4. Glycosylation

Human-compatible glycosylation can contribute to safety and efficacy inrecombinant drug products. Regulatory approval may be contingent ondemonstrating appropriate glycosylation as a critical quality attribute(see Zhang et al., Drug Discovery Today 21(5):740-765 (2016)). N-glycanscan result from glycosylation of asparagine side chains and can differin structure between humans and other organisms such as bacteria andyeast. Thus, it may be desirable to reduce or eliminate non-humanglycosylation and/or N-glycan formation in ferritin according to thedisclosure. In some embodiments, controlling glycosylation of ferritinimproves the efficacy and/or safety of the composition, especially whenused for human vaccination.

In some embodiments, ferritin is mutated to inhibit formation of anN-glycan. In some embodiments, a mutated ferritin has reducedglycosylation as compared to its corresponding wild type ferritin.

In some embodiments, the ferritin comprises a mutation replacing asurface-exposed asparagine with a non-asparagine amino acid. In someembodiments, the surface-exposed asparagine is N19 of H. pylori ferritinor a position that corresponds to position 31 of H. pylori ferritin asdetermined by pair-wise or structural alignment In some embodiments,mutating such an asparagine, e.g., N19 of H. pylori ferritin, decreasesglycosylation of ferritin. In some embodiments, the mutation replacesthe asparagine with a glutamine. In some embodiments, the ferritin is anH. pylori ferritin comprising an N19Q mutation. SEQ ID NOS: 201-207 areexemplary ferritin sequences comprising N19Q mutations.

A mammal exposed to a glycosylated protein produced in bacteria or yeastmay generate an immune response to the glycosylated protein, because thepattern of glycosylation of a given protein in bacterial or yeast couldbe different from the pattern of glycosylation of the same protein in amammal. Thus, some glycosylated therapeutic proteins may not beappropriate for production in bacteria or yeast.

In some embodiments, decreased glycosylation of ferritin by amino acidmutation facilitates protein production in bacteria or yeast. In someembodiments, decreased glycosylation of ferritin reduces the potentialfor adverse effects in mammals upon administration of mutated ferritinthat is expressed in bacteria or yeast. In some embodiments, thereactogenicity in a human subject of a mutated ferritin produced inbacteria or yeast is lower because glycosylation is decreased. In someembodiments, the incidence of hypersensitivity responses in humansubjects is lower following treatment with a mutated ferritin withreduced glycosylation compared to wildtype ferritin.

In some embodiments, degradation in a subject of a compositioncomprising a mutated ferritin with reduced glycosylation is slowercompared with a composition comprising a wild-type ferritin, or acomposition comprising a corresponding ferritin with wild-typeglycosylation. In some embodiments, a composition comprising a mutatedferritin with reduced glycosylation has reduced clearance in a subjectcompared with a composition comprising a wild-type ferritin, or acomposition comprising a corresponding ferritin with wild-typeglycosylation. In some embodiments, a composition comprising a mutatedferritin with reduced glycosylation has a longer-serum half-lifecompared to wild-type ferritin, or a composition comprising acorresponding ferritin with wild-type glycosylation.

5. Combinations of Mutations

In some embodiments, a ferritin comprises more than one type of mutationdescribed herein. In some embodiments, the ferritin comprises one ormore mutations independently selected from: a mutation to decreaseglycosylation, a mutation to remove an internal cysteine, and a mutationto generate a surface-exposed cysteine. In some embodiments, theferritin comprises a mutation to decrease glycosylation, a mutation toremove an internal cysteine, and a mutation to generate asurface-exposed cysteine.

In some embodiments, the ferritin comprises an N19Q mutation, a C31Smutation, and a mutation to generate a surface-exposed cysteine. In someembodiments, the ferritin comprises an N19Q mutation, a C31S mutation,and an E12C mutation. In some embodiments, the ferritin comprises anN19Q mutation, a C31S mutation, and an S72C mutation. In someembodiments, the ferritin comprises an N19Q mutation, a C31S mutation,and an A75C mutation. In some embodiments, the ferritin comprises anN19Q mutation, a C31S mutation, and an K79C mutation. In someembodiments, the ferritin comprises an N19Q mutation, a C31S mutation,and an S100C mutation. In some embodiments, the ferritin comprises anN19Q mutation, a C31S mutation, and an S111C mutation. In someembodiments, the ferritin comprises mutations corresponding to any ofthe foregoing sets of mutations, wherein the corresponding mutationschange an N to a Q, a C to an S, and a non-cysteine surface-exposedamino acid to a cysteine at positions determined by pair-wise alignmentof the ferritin amino acid sequence to an H. pylori ferritin amino acidsequence (SEQ ID NO: 208 OR 209).

Exemplary ferritins comprising more than one type of mutation areprovided in SEQ ID NOS: 201-207.

6. Structural Alignment

As discussed herein, positions of mutations corresponding to thosedescribed with respect to a given polypeptide (e.g, H. pylori ferritin)can be identified by pairwise or structural alignment. Structuralalignment is relevant to large protein families such as ferritin wherethe proteins share similar structures despite considerable sequencevariation and many members of the family have been structurallycharacterized, and can also be used to identify corresponding positionsin different versions of other polypeptides described herein, such asEBV polypeptides (e.g., gL, gH, gp220, or gp42). The protein databank(PDB) comprises 3D structures for many ferritins, including those listedbelow with their accession numbers.

2jd6, 2jd7-PfFR-Pyrococcus furiosus. 2jd8-PfFR+Zn. 3a68-soFR from geneSferH4-soybean. 3a9q-soFR from gene SferH4 (mutant). 3egm, 3bvf, 3bvi,3bvk, 3bv1-HpFR-Heliobacter pylori. 5c6f-HpFR (mutant)+Fe. 1z4a,1vlg-FR-Thermotoga maritime. 1s3q, 1sq3, 3kx9-FR-Archaeoglubus fulgidus.1krq-FR-Campylobacter jejuni. 1eum-EcFR-Escherichia coli. 4reu-EcFR+Fe.4xgs-EcFR (mutant)+Fe2O2. 4ztt-EcFR (mutant)+Fe2O+Fe2+Fe+O2.1qgh-LiFR-Listeria innocua. 3qz3-VcFR-Vibrio cholerae. 3vnx-FR-Ulvapertusa. 4ism, 4isp, 4itt, 4itw, 4iwj, 4iwk, 4ixk,3e6s-PnmFR-Pseudo-nitschia multiseries. 4zkh, 4zkw, 4zkx, 4zl5, 4zl6,4zlw, 4zmc-PnmFR (mutant)+Fe. 1z6o-FR-Trichoplusia ni.4cmy-FR+Fe-Chlorobaculum tepidum. Ferritin light chain (FTL). 1lb3,1h96-mFTL-mouse. 1rcc, 1rcd, 1rci-bFTL+tartrate+Mg. 1rce,1rcg-bFTL+tartrate+Mn. 3noz, 3np0, 3np2, 3o7r-hoFTL (mutant)-horse.3o7s, 3u90-hoFTL. 4v1w-hoFTL-cryo EM. 3rav, 3rd0-hoFTL+barbiturate.Ferritin light+heavy chains: 5gn8-hFTH+Ca.

Structural alignment involves identifying corresponding residues acrosstwo (or more) polypeptide sequences by (i) modeling the structure of afirst sequence using the known structure of the second sequence or (ii)comparing the structures of the first and second sequences where bothare known, and identifying the residue in the first sequence mostsimilarly positioned to a residue of interest in the second sequence.Corresponding residues are identified in some algorithms based onalpha-carbon distance minimization in the overlaid structures (e.g.,what set of paired alpha carbons provides a minimized root-mean-squaredeviation for the alignment). When identifying positions in a non-H.pylori ferritin corresponding to positions described with respect to H.pylori ferritin, H. pylori ferritin can be the “second” sequence. Wherea non-H. pylori ferritin of interest does not have an available knownstructure, but is more closely related to another non-H. pylori ferritinthat does have a known structure than to H. pylori ferritin, it may bemost effective to model the non-H. pylori ferritin of interest using theknown structure of the closely related non-H. pylori ferritin, and thencompare that model to the H. pylori ferritin structure to identify thedesired corresponding residue in the ferritin of interest. There is anextensive literature on structural modeling and alignment;representative disclosures include U.S. Pat. Nos. 6,859,736; 8,738,343;and those cited in Aslam et al., Electronic Journal of Biotechnology 20(2016) 9-13. For discussion of modeling a structure based on a knownrelated structure or structures, see, e.g., Bordoli et al., NatureProtocols 4 (2009) 1-13, and references cited therein.

7. Lumazine Synthase

In some embodiments, the antigenic polypeptide comprises a lumazinesynthase protein. Lumazine synthases can form higher-order structures,e.g., a 60-subunit lumazine synthase particle. Exemplary lumazinesynthases are Aquifex aeolicus lumazine synthase (SEQ ID NO: 40) and E.coli lumazine synthase (SEQ ID NO: 41). In some embodiments, thelumazine synthase has at least 85%, 90%, 95%, 97%, 98%, 99%, or 100%identity to the sequence of SEQ ID NOS: 40 or 41. The lumazine synthasecan be located C-terminal to the EBV polypeptide and can be separatedfrom the EBV polypeptide by a linker as discussed herein.

G. Mutations in gL, gH, Gp42, Linker, and/or Ferritin Sequences toEliminate Potential Oxidation, Deamidation, or Isoaspartate FormationSites

In some embodiments, an antigenic EBV polypeptide comprises one or moremutations to eliminate potential oxidation, deamidation, or Isoaspartateformation sites, such as the exemplary mutations set forth in Table 1below.

For example, in some embodiments, a gL sequence comprises one or moremutations to eliminate a potential succinimide/isoaspartate ordeamidation site. For example, a gL sequence can comprise a G to Amutation at a position corresponding to position 36 of SEQ ID NO: 227;an N to Q mutation at a position corresponding to position 47 of SEQ IDNO: 227; or an N to Q mutation at a position corresponding to position105 of SEQ ID NO: 227. A position in an amino acid sequence“corresponds” to a given position in SEQ ID NO: 227 if it aligns to thatposition according to a standard sequence alignment algorithm such asthe Smith-Waterman algorithm using default parameters.

In some embodiments, a linker comprises one or more mutations toeliminate a potential deamidation site. For example, a linker sequencecan comprise an N to G mutation at a position corresponding to position132 or 141 of SEQ ID NO: 227.

In some embodiments, a gH sequence comprises one or more mutations toeliminate a potential succinimide/isoaspartate or oxidation site. Forexample, a gH sequence can comprise an M to L mutation at a positioncorresponding to position 189, 401, or 729 of SEQ ID NO: 227; a D to Emutation at a position corresponding to position 368 of SEQ ID NO: 227;an M to I mutation at a position corresponding to position 499 or 639 ofSEQ ID NO: 227; or an N to Q mutation at a position corresponding toposition 653 of SEQ ID NO: 227.

In some embodiments, a gp42 sequence comprises one or more mutations toeliminate a potential deamidation site. For example, a gp42 sequence cancomprise an N to Q mutation at a position corresponding to position 959or 990 of SEQ ID NO: 227; or an N to S mutation at a positioncorresponding to position 988 of SEQ ID NO: 227.

In some embodiments, a ferritin sequence comprises one or more mutationsto eliminate a potential deamidation, oxidation, or isoaspartateformation site. For example, a ferritin sequence can comprise a Q to Smutation at a position corresponding to position 1150 of SEQ ID NO: 227;an M to I mutation at a position corresponding to position 1168 of SEQID NO: 227; an M to L mutation at a position corresponding to position1177 of SEQ ID NO: 227; a G to A mutation at a position corresponding toposition 1188 of SEQ ID NO: 227; or an N to Q mutation at a positioncorresponding to position 1253 or 1296 of SEQ ID NO: 227.

Exemplary mutations are shown below in Table 1. The position numberingcorresponds to SEQ ID NO: 227.

TABLE 1 Exemplary mutations. MO- solvent Location Modification START ENDTIF exposoure Mutation gL Succinimide/ 35 36 DG Exposed G35A IsoAsp gLdeamidation 47 47 N likely N47Q exposed gL deamidation 105 105 N exposedN105Q linker deamidation 132 132 N exposed N132G linker deamidation 141141 N exposed N141G gH oxidation 189 189 M exposed M189L gH Succinimide/368 369 DY exposed D368E IsoAsp gH oxidation 401 401 M buried M401L gHSuccinimide/ 429 430 DT exposed D429E IsoAsp gH oxidation 499 499 Mexposed M499I gH oxidation 639 639 M exposed M639I gH oxidation 653 653N exposed N653Q gH oxidation 729 729 M exposed M729L gp42 deamidation959 959 N exposed N959Q gp42 deamidation 988 988 N exposed N988S gp42deamidation 990 990 N exposed N990Q ferritin deamidation 1150 1150 Qexposed Q1150S ferritin oxidation 1168 1168 M buried M1168I ferritindeamidation 1177 1177 M buried M1177L ferritin IsoAsp 1187 1188 DGburied G1187A ferritin deamidation 1253 1253 N exposed N1253Q ferritindeamidation 1296 1296 N exposed N1296Q

H. Immune-Stimulatory Moieties; Adjuvants; Conjugated EBV Polypeptides

In some embodiments, an EBV polypeptide and/or an immune-stimulatorymoiety, such as an adjuvant, is attached to a surface-exposed aminoacid. In some embodiments, the surface-exposed amino acid is a cysteine,e.g., resulting from a mutation discussed above. In some embodiments,the surface-exposed amino acid is a lysine, aspartate, or glutamate.Conjugation procedures using glutaraldehyde (for conjugation of a lysinewith an amino-bearing linker or moiety) or a carbodiimide (e.g.,1-Cyclohexyl-3-(2-morpholin-4-yl-ethyl) carbodiimide or1-Ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC; EDAC) forconjugating an aspartate or glutamate to an amino-bearing linker ormoiety, or a lysine to a carboxyl-bearing linker or moiety) aredescribed in, e.g., Chapter 4 of Holtzhauer, M., Basic Methods for theBiochemical Lab, Springer 2006, ISBN 978-3-540-32785-1, available fromwww.springer.com.

In some embodiments, an immune-stimulatory moiety, such as an adjuvant,is attached to a surface-exposed amino acid of ferritin. In someembodiments, more than one immune-stimulatory moiety, such as anadjuvant, is attached to a surface-exposed amino acid of ferritin. Insome embodiments, twenty-four immune-stimulatory moieties are attachedto a ferritin multimer or particle (e.g., one moiety for each monomer inthe H. pylori ferritin particle). In some embodiments with multipleimmune-stimulatory moieties attached to a ferritin nanoparticle, all ofthe immune-stimulatory moieties are identical. In some embodiments withmultiple immune-stimulatory moieties attached to a ferritinnanoparticle, all of the immune-stimulatory moieties are not identical.

1. Types of Immune-Stimulatory Moieties; Adjuvants

Any immune-stimulatory moiety that can be attached to a surface-exposedamino acid (e.g., cysteine) can be used in ferritins according to thisdisclosure. In some embodiments, the immune-stimulatory moiety is a Bcell agonist.

In some embodiments, the immune-stimulatory moiety is not hydrophobic.In some embodiments, the immune-stimulatory moiety is hydrophilic. Insome embodiments, the immune-stimulatory moiety is polar. In someembodiments, the immune-stimulatory moiety is capable of hydrogenbonding or ionic bonding, e.g., comprises a hydrogen bond donor,hydrogen bond acceptor, cationic moiety, or anionic moiety. A moiety isconsidered cationic or anionic if it would be ionized in aqueoussolution at a physiologically relevant pH, such as pH 6, 7, 7.4, or 8.

In some embodiments, the immune-stimulatory moiety is an adjuvant. Insome embodiments, the adjuvant comprises a pathogen associated molecularpattern (PAMP). In some embodiments, the adjuvant is a toll-likereceptor (TLR) agonist or stimulator of interferon genes (STING)agonist. In some embodiments, the adjuvant activates TLR signaling in Band/or T cells. In some embodiments, the adjuvant regulates the adaptiveimmune response.

a) TLR2 Agonists

In some embodiments, the immune-stimulatory moiety is a TLR2 agonist. Insome embodiments, the immune-stimulatory moiety stimulates TLR2signaling. In some embodiments, the immune-stimulatory moiety is asynthetic small molecule ligand of TLR2. In some embodiments, theimmune-stimulatory moiety is a synthetic small molecule agonist of TLR2signaling.

In some embodiments, the TLR2 agonist is PAM2CSK4, FSL-1, or PAM3C SK4.

b) TLR7/8 Agonists

In some embodiments, the immune-stimulatory moiety is a TLR7 and/or TLR8agonist (i.e., an agonist of at least one of TLR7 and TLR8). In someembodiments, the immune-stimulatory moiety stimulates TLR7 and/or TLR8signaling. In some embodiments, the immune-stimulatory moiety is asynthetic small molecule ligand of TLR7 and/or TLR8. In someembodiments, the immune-stimulatory moiety is a synthetic small moleculeagonist of TLR7 and/or TLR8 signaling.

In some embodiments, the TLR7 and/or TLR8 agonist is single-stranded(ssRNA). In some embodiments, the TLR7 and/or TLR8 agonist is animidazoquinoline. In some embodiments, the TLR7 and/or TLR8 agonist is anucleoside analog.

In some embodiments, the TLR7 and/or TLR8 agonist is animidazoquinolinamine Toll-like receptor (TLR) agonist, such as 3M-012(3M Pharmaceuticals). The structure of free 3M-012 is:

It is understood that an immune-stimulatory moiety such as 3M-012 or anymoiety discussed herein can be conjugated to a ferritin by substitutingan appropriate peripheral atom of the moiety (e.g., a hydrogen) with abond to a ferritin described herein, e.g., at the sulfur of asurface-exposed cysteine or a linker attached to such a sulfur. Thus,when conjugated to a ferritin, the structure of the immune-stimulatorymoiety will differ slightly from the structure of the free molecule.

In some embodiments the TLR7 and/or TLR8 agonist is SM 7/8a. Thestructure of free SM 7/8a is:

See, e.g., Nat Biotechnol. 2015 November; 33(11):1201-10. doi:10.1038/nbt.3371.

c) TLR9 Agonists

In some embodiments, the immune-stimulatory moiety is a TLR9 agonist. Insome embodiments, the immune-stimulatory moiety stimulates TLR9signaling. In some embodiments, the immune-stimulatory moiety is asynthetic small molecule ligand of TLR9. In some embodiments, theimmune-stimulatory moiety is a synthetic small molecule agonist of TLR9signaling.

In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide(ODN). In some embodiments, the TLR9 agonist is an unmethylated CpG ODN.In some embodiments, the CpG ODN comprises a partial or completephosphorothioate (PS) backbone instead of the natural phosphodiester(PO) backbone found in ordinary DNA.

In some embodiments, the CpG ODN is a Class B ODN, which comprises oneor more 6mer CpG motif comprising 5′ Purine (Pu)-Pyrimidine(Py)-C-G-Py-Pu 3′; has a fully phosphorothioated (i.e., PS-modified)backbone; and has a length of 18-28 nucleotides. In some embodiments,the CpG ODN comprises the sequence of SEQ ID NO: 210, optionallycomprising phosphorothioate linkages in the backbone.

In some embodiments, the TLR9 agonist comprises an immune-stimulatorysequence (ISS). In some embodiments the TLR9 agonist is ISS-1018(Dynavax) (SEQ ID NO: 210).

d) STING Agonists

In some embodiments, the immune-stimulatory moiety is a STING(Stimulator of Interferon Genes Protein, also known as EndoplasmicReticulum IFN Stimulator) agonist. In some embodiments, theimmune-stimulatory moiety stimulates STING signaling. In someembodiments, the immune-stimulatory moiety is a synthetic small moleculeligand of STING. In some embodiments, the immune-stimulatory moiety is asynthetic small molecule agonist of STING signaling.

In some embodiments the STING agonist is a cyclic dinucleotide (CDN).See, e.g., Danilchanka et al., Cell 154:962-970 (2013). Exemplary CDNsinclude cdA, cdG, cAMP-cGMP, and 2′-5′,3′-5′ cGAMP (see Danilchanka etal. for structures). STING agonists also include synthetic agonists suchas DMXAA

2. Conjugated EBV Polypeptides

In some embodiments, an EBV polypeptide is conjugated to asurface-exposed amino acid of ferritin. In some embodiments, the EBVpolypeptide renders the ferritin protein antigenic. In some embodiments,the EBV polypeptide is antigenic alone, whereas in some embodiments, theEBV polypeptide is antigenic because of its association with ferritin.In some embodiments, the EBV polypeptide is any one of the EBVpolypeptides described herein.

3. Conjugation

In some embodiments, a surface-exposed cysteine (e.g., resulting from amutation described herein) or a cysteine in a peptide linker attached toferritin (e.g., N-terminally to ferritin) is used to conjugate animmune-stimulatory moiety, such as an adjuvant, or an EBV polypeptide toa ferritin. In some embodiments, a linker is conjugated to such acysteine, which linker can be subsequently conjugated to animmune-stimulatory moiety, such as an adjuvant, or an EBV polypeptide.In some embodiments, such a cysteine creates a chemical handle forconjugation reactions to attach an adjuvant, linker, or an EBVpolypeptide. In some embodiments, bioconjugates are produced, wherein animmune-stimulatory moiety, such as an adjuvant, or an EBV polypeptide islinked to a ferritin after reduction of such a cysteine. In someembodiments, the cysteine is an unpaired surface-exposed cysteine, i.e.,that lacks a partner cysteine in an appropriate position to form adisulfide bond. In some embodiments, the cysteine is an unpairedcysteine that comprises a free thiol side chain.

a) Types of Conjugation Chemistries

Any type chemistry can be used to conjugate the immune-stimulatorymoiety, such as an adjuvant, or an EBV polypeptide to the ferritin,e.g., via reaction a surface-exposed amino acid such as cysteine oranother amino acid such as Lys, Glu, or Asp.

In some embodiments, the conjugation is performed using click chemistry.As used herein, “click chemistry” refers to a reaction between a pair offunctional groups that rapidly and selective react (i.e., “click”) witheach other. In some embodiments, the click chemistry can be performedunder mild, aqueous conditions. In some embodiments, a click chemistryreaction takes advantage of a cysteine on the surface of the ferritin,such as a cysteine resulting from mutation of a surface-exposed aminoacid, to perform click chemistry using a functional group that can reactwith the cysteine.

A variety of reactions that fulfill the criteria for click chemistry areknown in the field, and one skilled in the art could use any one of anumber of published methodologies (see, e.g., Hein et al., Pharm Res25(10):2216-2230 (2008)). A wide range of commercially availablereagents for click chemistry could be used, such as those from SigmaAldrich, Jena Bioscience, or Lumiprobe. In some embodiments, conjugationis performed using click chemistry as described in the Examples below.

In some embodiments, the click chemistry reaction occurs after reductionof the ferritin.

In some embodiments, the click chemistry may be a 1-step click reaction.In some embodiments, the click chemistry may be a 2-step click reaction.

In some embodiments, the reaction(s) comprises metal-free clickchemistry. In some embodiments, the reaction(s) comprise thiol-maleimideand/or disulfide exchange.

Metal-Free Click Chemistry

Metal-free click chemistry can be used for conjugation reactions toavoid potential oxidation of proteins. Metal-free click chemistry hasbeen used to form antibody conjugates (see van Geel et al., BioconjugateChem. 2015, 26, 2233-2242).

In some embodiments, metal-free click chemistry is used in reactions toattach adjuvant to ferritin. In some embodiments, copper-freeconjugation is used in reactions to attach adjuvant to ferritin. In someembodiments, the metal-free click chemistry uses bicyclo[6.1.0]nonyne(BCN). In some embodiments, the metal-free click chemistry usesdibenzoazacyclooctyne (DBCO). In some embodiments BCN or DBCO reactswith an azide group.

DBCO has high specificity for azide groups via a strain-promoted clickreaction in the absence of a catalyst, resulting in high yield of astable triazole. In some embodiments, DBCO reacts with azide in theabsence of copper catalyst.

In some embodiments, metal-free click chemistry is used in a 1-stepclick reaction. In some embodiments, metal-free click chemistry is usedin a 2-step click reaction.

Thiol-Maleimide and Disulfide Exchange

Ferritins described herein can comprise a cysteine comprising a thiol,also known as a sulfhydryl, which is available for reaction withsulfhydryl-reactive chemical groups (or which can be made availablethrough reduction). Thus, the cysteine allows chemoselectivemodification to add an immune-stimulatory moiety, such as an adjuvant,to the ferritin. Under basic conditions, the cysteine will bedeprotonated to generate a thiolate nucleophile, which can react withsoft electrophiles, such as maleimides and iodoacetamides. The reactionof the cysteine with a maleimide or iodoacetamide results in acarbon-sulfur bond.

In some embodiments, a sulfhydryl-reactive chemical group reacts withthe surface-exposed cysteine or cysteine in the linker of the ferritin.In some embodiments, the sulfhydryl-reactive chemical group is ahaloacetyl, maleimide, aziridine, acryloyl, arylating agent,vinylsulfone, pyridyl disulfide, or TNB-thiol.

In some embodiments, the sulfhydryl-reactive chemical group conjugatesto the sulfhydryl of the cysteine by alkylation (i.e., formation of athioether bond)). In some embodiments, the sulfhydryl-reactive chemicalgroup conjugates to the sulfhydryl of the cysteine by disulfide exchange(i.e., formation of a disulfide bond).

In some embodiments, the reaction to conjugate an immune-stimulatorymoiety, such as an adjuvant, to the ferritin is a thiol-maleimidereaction.

In some embodiments, the sulfhydryl-reactive chemical group is amaleimide. In some embodiments, reaction of a maleimide with thecysteine results in formation of a stable thioester linkage, e.g., thatis not reversible. In some embodiments, the maleimide does not reactwith tyrosines, histidines, or methionines in the ferritin. In someembodiments, unreacted maleimides are quenched at the end of thereaction by adding a free thiol, e.g., in excess.

In some embodiments, the reaction to conjugate an immune-stimulatorymoiety, such as an adjuvant, to the ferritin is a thiol-disulfideexchange, also known as a disulfide interchange. In some embodiments,the reaction involves formation of a mixed disulfide comprising aportion of the original disulfide. In some embodiments, the originaldisulfide is the cysteine introduced in the ferritin by mutation of asurface-exposed amino acid or addition of an N-terminal linker.

In some embodiments, the sulfhydryl-reactive chemical group is a pyridyldithiol. In some embodiments, the sulfhydryl-reactive chemical group isa TNB-thiol group.

b) Linkers

In some embodiments, an immune-stimulatory moiety, such as an adjuvant,or an EBV polypeptide is attached to the ferritin via a linker that iscovalently bound to a surface-exposed amino acid such as a cysteine. Insome embodiments, the linker comprises a polyethylene glycol, e.g., aPEG linker. In some embodiments, the polyethylene glycol (e.g., PEG)linker increases water solubility and ligation efficiency of theferritin linked to the immune-stimulatory moiety, such as an adjuvant.The PEG linker is between 2 and 18 PEGs long, e.g., PEG4, PEG5, PEG6,PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16,PEG17, and PEG18.

In some embodiments, the linker comprises a maleimide. In someembodiments, the linker comprises the components of immune-stimulatorymoiety (ISM)-linker-maleimide. In some embodiments, theISM-linker-maleimide is conjugated to ferritin in a 1-step clickchemistry reaction by reaction of the maleimide with a cysteine of theferritin. In some embodiments, the ISM of the adjuvant-linker-maleimideis SM7/8a. In some embodiments, the linker of the ISM-linker-maleimideis PEG4. In some embodiments, the ISM-linker-maleimide isSM7/8a-PEG4-maleimide.

In some embodiments, a 2-step click chemistry protocol is used with alinker comprising a sulfhydryl-reactive chemical group at one end and anamine-reactive group at the other end. In such a 2-step click chemistryprotocol, a sulfhydryl-reactive chemical group reacts with a cysteine ofthe ferritin, while the amine-reactive group reacts with a reagentattached to the ISM. In this way, the ISM is conjugated to the ferritinvia a set of 2 click chemistry reagents.

In some embodiments of the 2-step click chemistry protocol, thesulfhydryl-reactive chemical group is maleimide. In some embodiments ofthe 2-step click chemistry protocol, the maleimide reacts with thecysteine introduced in the ferritin by mutation of a surface-exposedamino acid or addition of an N-terminal linker.

In some embodiments of the 2-step click chemistry protocol, theamine-reactive group is DBCO. In some embodiments of the 2-step clickchemistry protocol, the DBCO reacts with an azide group attached to anISM.

In some embodiments, a maleimide-linker-DBCO is used. In someembodiments, the maleimide-linker-DBCO is conjugated to ferritin afterthe ferritin is reduced. In some embodiments, themaleimide-linker-reagent is conjugated to ferritin by reaction of themaleimide with the cysteine of the ferritin in a first step. In someembodiments, the DBCO is used to link to an ISM attached to azide. Insome embodiments, the ISM coupled to azide is ISS-1018. In someembodiments, the adjuvant coupled to azide is 3M-012 or CpG.

In some embodiments, a linker with a reactive group is added to the ISM.In some embodiments, the linker is a PEG4-azide linker or aPEG4-maleimide linker.

In some embodiments, a PEG4-azide linker is conjugated to 3M-012. Anexemplary structure of 3M-012 conjugated to a PEG4-azide linker is:

In some embodiments, a PEG4-azide linker is conjugated to SM7/8a. Anexemplary structure of SM7/8a conjugated to a PEG4-azide linker is:

In some embodiments, a PEG4-maleimide linker is conjugated to SM7/8a. Anexemplary structure of SM7/8a conjugated to a PEG4-maleimide linker is:

In some embodiments, an azide group is conjugated to ISS-1018. Anexemplary structure of ISS-1018 conjugated to an NHS ester-azide linkeris:

I. Exemplary Compositions, Kits, Nucleic Acids, Uses, and Methods

In some embodiments, the present invention provides methods ofimmunizing a subject against infection with EBV. The present inventionfurther provides methods of eliciting an immune response against EBV ina subject. In some embodiments, the present methods compriseadministering to the subject an effective amount of a pharmaceuticalcomposition described herein to a subject. In some embodiments, thepresent methods comprises administering to the subject an effectiveamount of an antigenic EBV polypeptide or nanoparticle described hereinto a subject.

In some embodiments, a composition comprising any one or more of theantigenic EBV polypeptides described herein and a pharmaceuticallyacceptable vehicle, adjuvant, or excipient is provided.

In some embodiments, an antigenic EBV polypeptide, nanoparticle, orcomposition described herein is administered to a subject, such as ahuman or any of the subjects discussed below, to immunize againstinfection caused by EBV. In some embodiments, an antigenic EBVpolypeptide or nanoparticle described herein is administered to asubject, such as a human, to produce a protective immune response tofuture infection with EBV. In some embodiments, an antigenic EBVpolypeptide is administered. In some embodiments, an antigenic EBVpolypeptide comprising an EBV polypeptide and ferritin is administered,wherein the ferritin can have one or more mutations described herein. Insome embodiments, an antigenic EBV polypeptide or nanoparticlecomprising any one of SEQ ID NOS: 1-27 is administered.

In some embodiments, the protective immune response decreases theincidence of hospitalization. In some embodiments, the protective immuneresponse decreases the incidence of EBV infection, mononucleosis,complications caused by mononucleosis (e.g. hepatitis, encephalitis,severe hemolytic anemia, or splenomegaly), nasopharyngeal cancer,gastric cancer, or B lymphoma (e.g., Burkitt's or Hodgkin's lymphoma).

In some embodiments, a composition comprises one antigenic EBVpolypeptide (e.g., a monovalent composition). In some embodiments, acomposition comprises an antigenic EBV polypeptide comprising a gHpolypeptide. In some embodiments, a composition comprises an antigenicEBV polypeptide comprising a gL polypeptide. In some embodiments, acomposition comprises an antigenic EBV polypeptide comprising a gp220polypeptide.

In some embodiments, a composition comprises more than one antigenic EBVpolypeptide. In some embodiments, a composition comprises one or moreantigenic EBV polypeptides comprising more than one polypeptide encodedby EBV (i.e., a multivalent composition). In some embodiments, an EBVvaccine comprises nanoparticles comprising a gp220 polypeptide and,separately, nanoparticles comprising gH and gL polypeptides.

In some embodiments, any one or more of the antigenic EBV polypeptides,nanoparticles, or compositions described herein are provided for use inimmunizing against infection caused by EBV. In some embodiments, any oneor more of the polypeptides, nanoparticles, or compositions describedherein are provided for use in producing a protective immune response tofuture infection with EBV.

1. Subjects

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human.

In some embodiments, the subject is an adult (greater than or equal to18 years of age). In some embodiments, the subject is a child oradolescent (less than 18 years of age). In some embodiments, the subjectis elderly (greater than 60 years of age). In some embodiments, thesubject is a non-elderly adult (greater than or equal to 18 years of ageand less than or equal to 60 years of age).

In some embodiments, the composition is suitably formulated for anintended route of administration. Examples of suitable routes ofadministration include intramuscular, transcutaneous, subcutaneous,intranasal, oral, or transdermal.

In some embodiments, more than one administration of the composition isadministered to the subject. In some embodiments, a boosteradministration improves the immune response.

In some embodiments, any one or more of the antigenic polypeptides, orcompositions described herein are for use in a mammal, such as a primate(e.g., non-human primate, such as a monkey (e.g., a macaque, such asrhesus or cynomolgus) or ape), rodent (e.g., mouse or rat), ordomesticated mammal (e.g., dog, rabbit, cat, horse, sheep, cow, goat,camel, or donkey).

2. Adjuvants

An adjuvant may be administered together with the antigenic EBVpolypeptides and/or nanoparticles described herein to a subject, whereinadministration of such a combination may produce a higher titer ofantibodies against the EBV polypeptide(s) in the subject as compared toadministration of the EBV polypeptide(s) without the adjuvant. Anadjuvant may promote earlier, more potent, or more persistent immuneresponse to the EBV polypeptide(s).

In some embodiments, a composition comprises one adjuvant. In someembodiments, a composition comprises more than one adjuvant. In someembodiments, a composition does not comprise an adjuvant.

In some embodiments, an adjuvant comprises aluminum. In someembodiments, an adjuvant is aluminum phosphate. In some embodiments, anadjuvant is Alum (Alyhydrogel '85 2%; Brenntag—Cat #21645-51-2).

In some embodiments, an adjuvant is an organic adjuvant. In someembodiments, an adjuvant is an oil-based adjuvant. In some embodiments,an adjuvant comprises an oil-in-water nanoemulsion.

In some embodiments, an adjuvant comprises squalene. In someembodiments, the adjuvant comprising squalene is Ribi (Sigma adjuvantsystem Cat #S6322-1v1), Addavax™ MF59, AS03, or AF03 (see U.S. Pat. No.9,703,095). In some embodiments, the adjuvant comprising squalene is ananoemulsion.

In some embodiments, an adjuvant comprises a polyacrylic acid polymer(PAA). In some embodiments, the adjuvant comprising PAA is SPA09 (see WO2017218819).

In some embodiments, an adjuvant comprises non-metabolizable oils. Insome embodiments, the adjuvant is Incomplete Freund's Adjuvant (IFA).

In some embodiments, an adjuvant comprises non-metabolizable oils andkilled Mycobacterium tuberculosis. In some embodiments, the adjuvant isComplete Freund's Adjuvant (CFA).

In some embodiments, an adjuvant is a lipopolysaccharide. In someembodiments, an adjuvant is monophosphoryl A (MPL or MPLA).

3. Pharmaceutical Compositions

In various embodiments, a pharmaceutical composition comprising anantigenic EBV polypeptide described herein and/or related entities isprovided. In some embodiments, the pharmaceutical composition is animmunogenic composition (e.g., a vaccine) capable of eliciting an immuneresponse such as a protective immune response against a pathogen.

For example, in some embodiments, the pharmaceutical compositions maycomprise one or more of the following: (1) an antigenic EBV polypeptidecomprising an EBV polypeptide and a ferritin comprising a mutationreplacing a surface-exposed amino acid with a cysteine; (2) an antigenicEBV polypeptide comprising an EBV polypeptide and a ferritin comprisinga mutation replacing a surface exposed amino acid with a cysteine and animmune-stimulatory moiety linked to the cysteine; (3) an antigenic EBVpolypeptide comprising an EBV polypeptide and a ferritin comprising (i)a surface-exposed cysteine, (ii) a peptide linker N-terminal to theferritin protein, wherein the EBV polypeptide is N-terminal to thepeptide linker; (4) an antigenic EBV polypeptide comprising an EBVpolypeptide and a ferritin comprising (i) a mutation replacing a surfaceexposed amino acid with a cysteine and an immune-stimulatory moietylinked to the cysteine, (ii) a mutation replacing the internal cysteineat position 31 of H. pylori ferritin, or a mutation of an internalcysteine at a position that is analogous to position 31 of a non-H.pylori ferritin as determined by pair-wise or structural alignment, witha non-cysteine amino acid, and (iii) a mutation replacing asurface-exposed asparagine with a non-asparagine amino acid; or (5) aferritin particle comprising any of the foregoing polypeptides. In someembodiments, the pharmaceutical compositions may comprise an antigenicEBV gL/gH polypeptide, e.g., wherein the polypeptide comprises a linkerof at least 15 amino acids between the gL and gH polypeptide sequences.

In some embodiments, the present invention provides pharmaceuticalcompositions comprising antibodies or other agents related to theantigenic polypeptides described herein. In an embodiment, thepharmaceutical composition comprises antibodies that bind to and/orcompete with an antigenic polypeptide described herein. Alternatively,the antibodies may recognize viral particles or bacteria comprising thenon-ferritin polypeptide component of an antigenic polypeptide describedherein.

In some embodiments, the pharmaceutical compositions as described hereinare administered alone or in combination with one or more agents toenhance an immune response, e.g., an adjuvant described above. In someembodiments, a pharmaceutical composition further comprises an adjuvantdescribed above.

In some embodiments, the pharmaceutical composition further comprises apharmaceutically acceptable carrier or excipient. As used herein, theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich a pharmaceutical composition is administered. In exemplaryembodiments, carriers can include sterile liquids, such as, for example,water and oils, including oils of petroleum, animal, vegetable, orsynthetic origin, such as, for example, peanut oil, soybean oil, mineraloil, sesame oil and the like. In some embodiments, carriers are orinclude one or more solid components. Pharmaceutically acceptablecarriers can also include, but are not limited to, saline, bufferedsaline, dextrose, glycerol, ethanol, and combinations thereof. As usedherein, an excipient is any non-therapeutic agent that may be includedin a pharmaceutical composition, for example to provide or contribute toa desired consistency or stabilizing effect. Suitable pharmaceuticalexcipients include, but are not limited to, starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. In various embodiments,the pharmaceutical composition is sterile.

In some embodiments, the pharmaceutical composition contains minoramounts of wetting or emulsifying agents, or pH buffering agents. Insome embodiments, the pharmaceutical compositions of may include any ofa variety of additives, such as stabilizers, buffers, or preservatives.In addition, auxiliary, stabilizing, thickening, lubricating, andcoloring agents can be included.

In various embodiments, the pharmaceutical composition may be formulatedto suit any desired mode of administration. For example, thepharmaceutical composition can take the form of solutions, suspensions,emulsion, drops, tablets, pills, pellets, capsules, capsules containingliquids, gelatin capsules, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, lyophilizedpowder, frozen suspension, desiccated powder, or any other form suitablefor use. General considerations in the formulation and manufacture ofpharmaceutical agents may be found, for example, in Remington'sPharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, Pa.,1995; incorporated herein by reference.

The pharmaceutical composition can be administered via any route ofadministration. Routes of administration include, for example, oral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, mucosal, epidural, sublingual, intranasal, intracerebral,intravaginal, transdermal, rectally, by intratracheal instillation,bronchial instillation, inhalation, or topically. Administration can belocal or systemic. In some embodiments, administration is carried outorally. In another embodiment, the administration is by parenteralinjection. In some instances, administration results in the release ofthe antigenic ferritin polypeptide described herein into thebloodstream. The mode of administration can be left to the discretion ofthe practitioner.

In some embodiments, the pharmaceutical composition is suitable forparenteral administration (e.g. intravenous, intramuscular,intraperitoneal, and subcutaneous). Such compositions can be formulatedas, for example, solutions, suspensions, dispersions, emulsions, and thelike. They may also be manufactured in the form of sterile solidcompositions (e.g. lyophilized composition), which can be dissolved orsuspended in sterile injectable medium immediately before use. Forexample, parenteral administration can be achieved by injection. In suchembodiments, injectables are prepared in conventional forms, i.e.,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions. Insome embodiments, injection solutions and suspensions are prepared fromsterile powders, lyophilized powders, or granules.

In a further embodiment, the pharmaceutical composition is formulatedfor delivery by inhalation (e.g., for direct delivery to the lungs andthe respiratory system). For example, the composition may take the formof a nasal spray or any other known aerosol formulation. In someembodiments, preparations for inhaled or aerosol delivery comprise aplurality of particles. In some embodiments, such preparations can havea mean particle size of about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, orabout 13 microns. In some embodiments, preparations for inhaled oraerosol delivery are formulated as a dry powder. In some embodiments,preparations for inhaled or aerosol delivery are formulated as a wetpowder, for example through inclusion of a wetting agent. In someembodiments, the wetting agent is selected from the group consisting ofwater, saline, or other liquid of physiological pH.

In some embodiments, the pharmaceutical composition in accordance withthe invention are administered as drops to the nasal or buccal cavity.In some embodiments, a dose may comprise a plurality of drops (e.g.,1-100, 1-50, 1-20, 1-10, 1-5, etc.).

The present pharmaceutical composition may be administered in any doseappropriate to achieve a desired outcome. In some embodiments, thedesired outcome is the induction of a long-lasting adaptive immuneresponse against a pathogen, such as the source of a non-ferritinpolypeptide present in an antigenic ferritin polypeptide present in thecomposition. In some embodiments, the desired outcome is a reduction inthe intensity, severity, frequency, and/or delay of onset of one or moresymptoms of infection. In some embodiments, the desired outcome is theinhibition or prevention of infection. The dose required will vary fromsubject to subject depending on the species, age, weight, and generalcondition of the subject, the severity of the infection being preventedor treated, the particular composition being used, and its mode ofadministration.

In some embodiments, pharmaceutical compositions in accordance with theinvention are administered in single or multiple doses. In someembodiments, the pharmaceutical compositions are administered inmultiple doses administered on different days (e.g., prime-boostvaccination strategies). In some embodiments, the pharmaceuticalcomposition is administered as part of a booster regimen.

In various embodiments, the pharmaceutical composition isco-administered with one or more additional therapeutic agents.Co-administration does not require the therapeutic agents to beadministered simultaneously, if the timing of their administration issuch that the pharmacological activities of the additional therapeuticagent and the active ingredient(s) in the pharmaceutical compositionoverlap in time, thereby exerting a combined therapeutic effect. Ingeneral, each agent will be administered at a dose and on a timeschedule determined for that agent.

4. Nucleic Acid/mRNA

Also provided is a nucleic acid encoding an antigenic EBV polypeptidedescribed herein. In some embodiments, the nucleic acid is an mRNA. Anynucleic acid capable of undergoing translation resulting in apolypeptide is considered an mRNA for purposes of this disclosure.

5. Kits

Also provided herein are kits comprising one or more antigenic EBVpolypeptides, nucleic acids, antigenic ferritin particles, antigeniclumazine synthase particles, compositions, or pharmaceuticalcompositions described herein. In some embodiments, a kit furthercomprises one or more of a solvent, solution, buffer, instructions, ordesiccant.

This description and exemplary embodiments should not be taken aslimiting. For the purposes of this specification and appended claims,unless otherwise indicated, all numbers expressing quantities,percentages, or proportions, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about,” to the extent they are not already somodified. “About” indicates a degree of variation that does notsubstantially affect the properties of the described subject matter,e.g., within 10%, 5%, 2%, or 1%. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained. At the veryleast, and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed considering the number of reported significantdigits and by applying ordinary rounding techniques.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” and any singular use of anyword, include plural referents unless expressly and unequivocallylimited to one referent. As used herein, the term “include” and itsgrammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items. The term“or” is used in the inclusive sense, i.e., equivalent to “and/or,”unless the context dictates otherwise.

TABLE 2 (SEQUENCE TABLE): DESCRIPTION OF THE SEQUENCES SEQ IDDescription Sequences NO SIB 7187 leader sequence gp220 bfpFerrNanoparticle N19Q/C31S/S111C

  1 SIB 7340 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA   2leader sequence NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRGGSGSASSGASASGSSNGSGSGSGSNSSAS gL SGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSG(D7)_linker_gHTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALbfpFerrSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNNanoparticleYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKN19Q/C31S/S111CSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTT

SIB 7342 METDTLLLWVLLLWVPGSTGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANG   3leader sequence LNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRGGGSGGGSGGGSGGGSG AASLSEVKLHLDIEG gLHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGV(D7)_linker_gHISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEbfpFerrHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLNanoparticleLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLN19Q/C31S/S111CAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTT

SIB 7379 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA   4leader sequence NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRGGSGSASSGASASGSSNGSGSGSGSNSSAS gL SGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSG(D7)_linker_gHTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALTrimerSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGSGSGLVPRGSGAGGGHHHHHH SIB 7380 METDTLLLWVLLLWVPGSTGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANG   5leader sequence LNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRGGGSGGGSGGGSGGGSG AASLSEVKLHLDIEG gLHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGV(D7)_linker_gHISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDETrimerHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTT

SIB 7381 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA 6leader sequence NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRGGSGSASSGASASGSSNGSGSGSGSNSSAS gL SGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSG(D7)_linker_gHTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALMonomerSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTT

SIB 7382 METDTLLLWVLLLWVPGSTGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANG   7leader sequence LNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRGGGSGGGSGGGSGGGSG AASLSEVKLHLDIEG gLHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGV(D7)_linker_gHISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEMonomerHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTT

SIB 7392 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNCLVISRCA   8leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gL_linker_gH GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK MonomerLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEK

SIB 7397 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNCLVISRCA   9leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSGSNSSASSGASSGGASGG gL_linker_gH SGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDMonomerIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSI

SIB 7400 METDTLLLWVLLLWVPGSTGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNCLVISRCANG  10leader sequence LNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGGGSGGGGSGGGGSG AASLSEVKL gL_linker_gHHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASbfpFerrKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVNanoparticleTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVN19Q/C31S/S111CLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVH

SIB 7402 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA  11leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSGSNSSASSGASSGGASGG gL_linker_gH_ SGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDtrimerIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSI

SIB 7403 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA  12leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gL_linker_gH_ GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK trimerLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEK

SIB 7404 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA  13leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSGSNSSASSGASSGGASGG gL_linker_gH SGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDbfpFerrIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGNanoparticleAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSN19Q/C31S/S111CRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSI

SIB 7406 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA  14leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gL_linker_gH GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK bfpFerrLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCNanoparticleHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVN19Q/C31S/S111CHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEK

SIB 7414 METDTLLLWVLLLWVPGSTGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANG  15leader sequence LNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGGGSGGGGSGGGGSG AASLSEVKL gL_linker_gHHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASMonomerKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVH

SIB 7429 METDTLLLWVLLLWVPGSTGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANG  16leader sequence LNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGGGSGGGGSGGGGSG AASLSEVKL gL_linker_gH_HLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDAStrimerKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVH

SIB 15000 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA  17leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASAEAAAKEAAAKAGGSG gL_FR1_gH GSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPbfpFerrAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMNanoparticleTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAN19Q/C31S/S111CVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILS

SIB 15001 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA  18leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASAEAAAKEAAAKEAAAK gL_FR2_gH ASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAbfpFerrEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFNanoparticleQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKN19Q/C31S/S111CDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEV

SIB 15002 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA  19leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS Construct 5 GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK gL_linker_gH_LIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRC linker bfpFerrHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVNanoparticleHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVN19Q/C31S/S111CDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSGASS

SIB 15003 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNCLVISRCA  20leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS Construct 7 GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK gL_linker_gH_LIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRClinker bfpFerrHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVNanoparticleHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVN19Q/C31SDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSCSGSGSGSSSASSGASS

SIB 15004 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNCLVISRCA  21leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gL_gH_gp42_  GSNSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK bfpFerr LIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCNanoparticleHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVN19Q/C31S/S111CHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSG DSKGSSQKGSRLLLLLVVSNLLLPQGVLAYFLPPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNFNKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTFKVYQIFGSHCTYVSKFSTVPVSHHECSFLKPCLCVSQRSNSGGSGSASSGASASG SSGSGSGSGSSSASSGASSGGASGG

SIB 15005 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA  22leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS Construct 5 GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK gL_gH_C137A_LIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRAbfpFerrHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVNanoparticle HYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIN19Q/C31S/S111CGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSGASS

SIB 15006 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA  23leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS Construct 7 GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK gL_gH_C137A_LIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRAbfpFerr HLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVNanoparticleHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIN19Q/C31SGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSCSGSGSGSSSASSGASS

SIB 17395 leader sequence gp220-T. ni ferritin heavy chain

 24 SIB 17396 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA  25leader sequenceNGFLNVVSFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gL_linker_gH- GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK T. ni ferritinLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCheavy chainHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEK

SIB 17397 leader sequence gp220-T. ni ferritin light chain

 26 SIB 17398 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA  27leader sequenceNGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gL_linker_gH- GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYK T. ni ferritinLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRClight chainHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEK

16 amino acid GGGGSGGGGSGGGGSG  28 linker 28 amino acidGGSGSGSNSSASSGASSGGASGGSGGSG  29 linker 46 amino acidGGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGSGGSG  30 linker FR1GGSGSASAEAAAKEAAAKAGGSGGSG  31 FR2 GGSGSASAEAAAKEAAAKEAAAKASGGSGGSG  3247 amino acid SGGGSGSASSGASASGSSCSGSGSGSSSASSGASSGGASGGGSGGSG  33 linkercomprising a C for conjugation Gp42DSKGSSQKGSRLLLLLVVSNLLLPQGVLAYFLPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNFNKTAEQEYGDKE 34TVKLPHWTPLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTFKVYQIFGSHCTYVSKFSTVPVSHHECSFLKPCLCVSQRSNS CpGT*G*A*C*T*G*T*G*A*A*C*G*T*T*C*G*A*G*A*T*G*A  35 (phosphorothi- oatemodifications where * is shown) Exemplary gLNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCANGLNVVSFFISILKRSSSALTG 36 polypeptide HLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGG Exemplary gHAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVS 37 polypeptideEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERA Exemplary gp220EAALLVCQYTIQSLIHLTGEDPGFFNVEIPEFPFYPTCNVCTADVNVTINFDVGGKKHQLDLDFGQLTPHTKAVYQPRGA 38 polypeptideFGGSENATNLFLLELLGAGELALTMRSKKLPINVTTGEEQQVSLESVDVYFQDVFGTMWCHHAEMQNPVYLIPETVPYIKWDNCNSTNITAVVRAQGLDVTLPLSLPTSAQDSNFSVKTEMLGNEIDIECIMEDGEISQVLPGDNKFNITCSGYESHVPSGGILTSTSPVATPIPGTGYAYSLRLTPRPVSRFLGNNSILYVFYSGNGPKASGGDYCIQSNIVFSDEIPASQDMPTNTTDITYVGDNATYSVPMVTSEDANSPNVTVTAFWAWPNNTETDFKCKWTLTSGTPSGCENISGAFASNRTFDITVSGLGTAPKTLIITRTATNATTTTHKVIFSKAPE Cysteine- CLVPRGSLEHHHHHH  39 Thrombin-HisLinker LumazineMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGV 40 synthase ofLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRAquifex aeolicus (strain VF5) E. coli 6,7-MNIIEANVATPDARVAITIARFNNFINDSLLEGAIDALKRIGQVKDENITVVWVPGAYELPLAAGALAKTGKYDAVIALG 41 dimethyl-8-TVIRGGTAHFEYVAGGASNGLAHVAQDSEIPVAFGVLTTESIEQAIERAGTKAGNKGAEAALTALEMINVLKAIKAribityllumazine synthase Not Used  42- 200 bfpFerritin-ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMCMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS201 N19Q/C31S/S26CAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS bfpFerritin-ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTCIS202 N19Q/C31S/S72CLAPEHKFEGTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS bfpFerritin-ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS203 N19Q/C31S/A75CCPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS bfpFerritin-ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS204 N19Q/C31S/K79CAPEHCFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS bfpFerritin-ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS205 N19Q/C31S/S100CAPEHKFEGLTQIFQKAYEHEQHISECINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS bfpFerritin-ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS206 N19Q/C31S/S111CAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKCKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS bfpFerritin-ESQVRQQFSKDIEKLLNCQVNKEMQSSNLYMSMSSWSYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS207 N19Q/C31S/E12CAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Exemplary H.ESQVRQQFSKDIEKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSIS208 pylori FerritinAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLAwith bullfrog DQYVKGIAKSRKS linker Exemplary wild-LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFE209 type H. pyloriGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIferritin AKSRKS (GenBank Accession AAD06160.1) (without bullfrog linkeror N-terminal Met) CpG (ISS-1018) TGACTGTGAACGTTCGAGATGA 210Trichoplusia niTQCNVNPVQIPKDWITMHRSCRNSMRQQIQMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLM211 heavy chainRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLferritin AGKASTLKKLMDRHEALGEF IFDKKLLGIDV Trichoplusia niADTCYNDVALDCGITSNSLALPRCNAVYGEYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLS212 light chainDEAWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAferritin QYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTV PyrococcusMLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNY 213furiosus IYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLferritin EWFINEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGEhuman heavyMTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGR214 chain ferritinIFLQDIKKPDCDDWESGLNAMECALHLEKNVQQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDQES human lightMDSKGSSQKGSRLLLLLVVSNLLLPQGVLASSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHF215 chain ferritinFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLE(signal peptide THFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHDis underlined) lumazineMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGV216 synthase fromLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRAquifex aeolicus bullfrog linker ESQVRQQF 217 Cysteine- CLVPRGSLEHHHHHH218 Thrombin-His Linker E. coli 6,7-MNITEANVATPDARVAITIARFNNFINDSLLEGAIDALKRIGQVKDENITVVWVPGAYELPLAAGALAKTGKYDAVIALG219 dimethy1-8-TVIRGGTAHFEYVAGGASNGLAHVAQDSEIPVAFGVLTTESIEQATERAGTKAGNKGAEAALTALEMINVLKAIKAribityllumazine synthase 16 amino acid GGGGSGGGGSGGGGSG 220 linker28 amino acid GGSGSGSNSSASSGASSGGASGGSGGSG 221 linker 46 amino acidGGSGSASSGASASGSSNGSGSGSGSNSSASSGASSGGASGGSGGSG 222 linker FR1GGSGSASAEAAAKEAAAKAGGSGGSG 223 FR2 GGSGSASAEAAAKEAAAKEAAAKASGGSGGSG 22447 amino acid SGGGSGSASSGASASGSSCSGSGSGSSSASSGASSGGASGGGSGGSG 225 linkercomprising a C for conjugation SIB 15007 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNCLVISRCA 226gL/gH/gp42-His NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRAHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDEDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSG LAYFLPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTESYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTEKVYQI F GSHCTYVSK F STVPVSHHECSFLKPCLCVSQRSNSGSHHHHHH SIB 15008MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA 227gH/gL/gp42 NP NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRAHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDEDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSG LAYFLPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTESYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTEKVYQIFGSHCTYVSKFSTVPVSHHECSFLKPCLCVSQRSNSGGSGASSGASASGSSGSGSGSGSSSASSGASSGGASGGS GGSGGGSGASSGASASGSSGSGSGS

SIB 15009 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA 228 gH/gL/NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gp42_NP_C12 GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRAHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVEGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMESRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLELSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDEDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSG LAYFLPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTEKVYQIFGSHCTYVSKFSTV

SIB 15010 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA 229 gH/gL/NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gp42_NP_C13 GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVEYQLRAHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVEGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMESRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLELSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSG LAYFLPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTEKVYQIFGSHCTYVSKFSTV

SIB 15011 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDGESLASLNSPKNGSNQLVISRCA 230 gH/gL/NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS gp42_NP_C14 GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVEYQLRAHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLELSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDEDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSSSASSG LAYFLPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPCNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTEKVYQIFGSHCTYVSKFSTV

SIB 15012 MDSKGSSQKGSRLLLLLVVSNLLLPQGVLANWAYPCCHVTQLRACHLLALENISDIYLVSNQTCDAFSLASLNSPKGGSN 231 gH/gL/CLVISRCANGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGAGLNRYAWHRGGGGSGSASSGASASG gp42_NP_C16 SGGSGSGSGSGSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELLAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVEYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGEYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELETETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAILMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSIAPQEATLDQAAVSQAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLELSPVILNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDEDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSS SASSGAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTFSYKGCCFYFTKKKHTWQGCFQACAELYPCTYFYGPTPDILPVVTRSLQAIESLWVGVYRVGEGNWTSLDGGTEKVYQIFGSHCTYVSKFSTVPVSHHECSFLKPCLCVSQRSNSGGSGSASSGASASGSSGSGSGSGSSSASSGASSGGASGGSGGSGGGSGSASSGASASGSSGSGSGSGSSSASSGAS

SIB 15013 MRAVGVFLAICLVTIFVLPTWGNWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDGFSLASLNSPKNGSNQLVISRCA 232gH/gL_rigid_NP NGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGANLNRYAWHRGGGGSGSASSGASASGSSNGSGSGS GSNSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELMAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRAHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGDYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELDTETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTTYFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTITSQEVQNSILSSNYFDEDNLHVHYLLLTTNGTVMEIAGLYEERASGEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPE

46 amino acid GGSGASSGASASGSSGGSGSGSGSGSSASSGASSGGASGGSGGSG 233 linker32 amino acid SGGGSGSASSGASASGSSGSGSGSGSSSASSG 234 linker 88 amino acidGGSGSASSGASASGSSGSGSGSGSSSASSGASSGGASGGSGGSGGGSGSASSGASASGSSGSGSGSGSSSASSGASSGG235 linker ASGGSGGSG SGGSGGSG 44 amino acidGGSGSASSGASASGSSGSGSGSGSSSASSGASSGGASGGSGGSG 236 linker 12 amino acidEPEPEPEPEPGG 237 rigid linker 48 amino acidSGEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEP 238 rigid linkergp42 fusionLAYFLPPRVRGGGRVAAAAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYC239 segmentNTREYTESYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTESTVPVSHHECSFLKPCLCVSQRSNS gp42 fusionAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTESYKGCCFYFTK240 segment 2KKHTWQGCFQACAELYPCTYFYGPTPDILPVVTRSLQAIESLWVGVYRVGEGNWTSLDGGTFKVYQIFGSHCTYVSKESTVPVSHHECSFLKPCLCKVYQIFGSHCTYVSKFVSQRSNS SIB 15014MDSKGSSQKGSRLLLLLVVSNLLLPQGVLANWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDAFSLASLNSPKQGSN 241 gH/gL/QLVISRCANGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGAQLNRYAWHRGGGGSGSASSGASASGS gp42_NP_C17 SGGSGSGSGSGSSASSNPGASSGGASGGAGGAGAASLSEVKLHLDIEGHASHYTIPWTELLAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGEYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELETETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAILMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSIAPQEATLDQAAVSQAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSG SASSGAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTESYKGCCFYFTKKKHTWQGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTEKVYQIFGSHCTYVSKESTVPVSHHECSFLKPCLCVSQRSNSGGSGSASSGASASGSSGSGSGSGSSSASSGASSGGASGGSGGSGGGSGSASSGASASGSSGSGSGSGSSSASSGASSSGASASGSSGSGSGSGSSSASSGAS

SIB 15015 MDSKGSSQKGSRLLLLLVVSNLLLPQGVLANWAYPCCHVTQLRAQHLLALENISDIYLVSNQTCDAFSLASLNSPKQGSN 242 gH/QLVISRCANGLNVVSFFISILKRSSSALTGHLRELLTTLETLYGSFSVEDLFGAQLNRYAWHRGGGGSGSASSGASASGS gL/gp42_NP_c18 SGGSGSGSGSGSSASSGASSGGASGGSGGSGAASLSEVKLHLDIEGHASHYTIPWTELLAKVPGLSPEALWREANVTEDLASMLNRYKLIYKTSGTLGIALAEPVDIPAVSEGSMQVDASKVHPGVISGLNSPACMLSAPLEKQLFYYIGTMLPNTRPHSYVFYQLRCHLSYVALSINGDKFQYTGAMTSKFLMGTYKRVTEKGDEHVLSLVFGKTKDLPDLRGPFSYPSLTSAQSGEYSLVIVTTFVHYANFHNYFVPNLKDMFSRAVTMTAASYARYVLQKLVLLEMKGGCREPELETETLTTMFEVSVAFFKVGHAVGETGNGCVDLRWLAKSFFELTVLKDIIGICYGATVKGMQSYGLERLAAMLMATVKMEELGHLTTEKQEYALRLATVGYPKAGVYSGLIGGATSVLLSAYNRHPLFQPLHTVMRETLFIGSHVVLRELRLNVTTQGPNLALYQLLSTALCSALEIGEVLRGLALGTESGLFSPCYLSLRFDLTRDKLLSMAPQEATLDQAAVSNAVDGFLGRLSLEREDRDAWHLPAYKCVDRLDKVLMIIPLINVTFIISSDREVRGSALYEASTTYLSSSLFLSPVIMNKCSQGAVAGEPRQIPKIQNFTRTQKSCIFCGFALLSYDEKEGLETTTYITSQEVQNSILSSNYFDFDNLHVHYLLLTTNGTVMEIAGLYEERASGGGSGSASSGASASGSSGSGSGSGSS SASSGAITWVPKPNVEVWPVDPPPPVNENKTAEQEYGDKEVKLPHWTPTLHTFQVPQNYTKANCTYCNTREYTESYKGCCFYFTKKKHTWNGCFQACAELYPCTYFYGPTPDILPVVTRNLNAIESLWVGVYRVGEGNWTSLDGGTEKVYQIFGSHCTYVSKESTVPVSHHECSFLKPCLCVSQRSNSGGSGSASSGASASGSSGSGSGSGSSSASSGASSGGASGGSGGSGGGSGSASSGASASGSSGSGSGSSSASSGASGASASGSSGSGSGSGSSSASSGAS

Key for SEQ ID NOs: 1-41 Leader Sequence — underlined gL — ItalicizedLinker — double underlined gH — Bold bfpFerr (ferritin) — wavy underlineFR — Italicized and double underline gp220 — Italicized and bold gp42— Italicized and underlined T. ni ferritin heavy chain — double wavyunderline Foldon sequence: Italicized and wavy underline Thrombincleavage site: Italicized and dashed underline 6X His Tag (SEQ ID NO:243): Bold, italicized and curvy underline

Examples

The following examples are provided to illustrate certain disclosedembodiments and are not to be construed as limiting the scope of thisdisclosure in any way.

1. Antigenic EBV Polypeptides for Eliciting Antibodies Against EBV

Antigenic polypeptides that elicit antibodies against EBV weredeveloped. Self-assembling ferritin nanoparticles were developed thatdisplay EBV gT and gH polypeptides as a single-chain, and theimmunogenicity of these nanoparticles in mice was evaluated.

Monomeric and trimeric gL/gH constructs were expressed and purified.FIG. 1A shows single-chain gL and gH monomer (SEQ ID NO: 6)+/−His-tagcleavage by Coomassie and western blot (anti-His) analysis. FIG. 1Bshows fractionation of a gL and gH trimer (SEQ ID NO: 11) on a Superose®SEC column as an absorbance trace and by Coomassie, along with a westernblot to confirm His-tag cleavage by thrombin protease. The finalconcentration of samples was 1 mg/mL, the total volume was 15 mL, andthe endotoxin level was 1.48 EU/mL for the SEQ ID NO: 6 construct.

Single-chain gL/gH ferritin nanoparticles (SEQ ID NO: 14) were expressedand purified. FIGS. 2A-2E show purification and characterization thereofby Superose® 6 SEC fractionation (2A), Coomassie of SEC fractions (2B),western blot of SEC fractions with anti-ferritin primary Ab (2C),dynamic light scattering (DLS, 2D), and electron microscopy (2E).

Exemplary constructs of single-chain EBV gL and gH fused to ferritin areshown in FIG. 3 . A conjugation site for an immune-stimulatory moiety,such as a toll-like receptor 7/8 agonist (TLR7/8a), can be presenteither on the ferritin or in the linker (see, e.g., SEQ ID NOS: 14, 19,22, 20, 23, and 33 for exemplary sequences).

gL/gH trimers or nanoparticles with different linkers were injected intomice and immune sera were assessed (FIG. 4 ). Mice were given two 2-μginjections with adjuvant AF03, a squalene emulsion-based adjuvant, witha 3-week interval between doses. Anti-gL/gH antibody endpoint titerswere measured by ELISA at week 6. For gH_16_gL, a nanoparticle (SEQ IDNO: 10) outperformed a trimer construct (SEQ ID NO: 16). The gL_28_gHnanoparticle (SEQ ID NO: 13) did not perform significantly differentlyfrom the trimer construct (SEQ ID NO: 11). The gL_46_gH nanoparticle(SEQ ID NO: 14) outperformed the gL_46_gH trimer (SEQ ID NO: 12).

These data indicate that single-chain gL/gH nanoparticles can elicit arobust immune response against EBV.

2. Bivalent Immunization Against gL/gH and Gp220

Bivalent immunization was performed using compositions comprisingsingle-chain gL/gH nanoparticles and gp220 nanoparticles. Including thegp220 nanoparticles (SEQ ID NO: 1) had no significant interfering effecton the immune response elicited by single-chain gL/gH nanoparticles(gL-gH_C5 NP [SEQ ID NO: 19]), as measured by an ELISA binding assayusing sera from mice vaccinated as described above (FIGS. 5A-5B, showingmeasurements at individual dilutions and binding titers, respectively).Similarly, no interference was observed in the response to the immuneresponse to gp220 nanoparticles when administered in combination withthe single-chain gL/gH nanoparticles, as measured by ELISA (FIGS. 6A-6B,showing measurements at individual dilutions and binding titers,respectively).

Thus, immunization with both a single-chain gL/gH nanoparticle and agp220 nanoparticle did not decrease the immune response to eitherpolypeptide.

3. Conjugation of Adjuvant to Ferritin Nanoparticles

Next, conjugation of adjuvants to ferritin nanoparticles was assessed.FIG. 7A illustrates a construct in which the ferritin comprises amutation replacing a surface-exposed amino acid with a cysteine, whichis available for conjugation. FIG. 7B shows an exemplaryimmune-stimulatory moiety (SM7/8a, a TLR-7/8 agonist) linked to a PEG4linker and maleimide. This maleimide can be used to covalently conjugatethe linker (itself attached to SM7/8a) to the surface-exposed cysteineof the ferritin. A polypeptide comprising a single-chain gL/gHpolypeptide fused to ferritin conjugated to SM7/8a is shown in theelectron micrograph of FIG. 7C.

A cysteine resulting from mutation of a surface-exposed amino acid isillustrated in the structure a ferritin molecule in FIG. 8A. Conjugationof a CpG adjuvant (SEQ ID NO: 230) to ferritin is illustrated in FIG. 8Bby juxtaposing the ferritin, linker, and CpG adjuvant, oriented to showthe parts of each moiety that become attached to each other inproximity.

A gL/gH nanoparticle (SEQ ID NO: 19) was reduced using 2 mM TCEP andthen oxidized via the addition of 1×PBS and using a 100 kD microspincolumn to remove TCEP. SM7/8a was then incubated with the gL/gHnanoparticle for conjugation. Excess SM7/8a was removed from thereaction via a 100 kD microspin column. Mass spectrometry (MS) dataindicated that about 100% of the polypeptide comprising single-chaingL/gH and ferritin (SEQ ID NO: 19) was conjugated to SM7/8a (FIG. 9B)based on shift of the main MS peak relative to the spectrum of theunconjugated polypeptide (FIG. 9A). The difference between the mass ofthe conjugated and unconjugated polypeptide corresponds to the molecularweight of the SM7/8a-linker-maleimide adduct (711 Da).

A gp220 nanoparticle (SEQ ID NO: 1) was reduced using 2 mM TCEP and thenoxidized via the addition of 1×PBS and using a 100 kD microspin columnto remove TCEP. The SM7/8a was then incubated with the gL/gHnanoparticle for conjugation. Excess SM7/8a was removed from thereaction via a 100 kD microspin column. MS data indicated that about100% of a conjugated polypeptide comprising gp220 and ferritin (SEQ IDNO: 1) is conjugated to SM7/8a (FIG. 10B) based on shift of the main MSpeak relative to the spectrum of the unconjugated polypeptide (FIG.10A).

Electron microscopy (EM) data also confirmed that conjugation of SM7/8ato polypeptides comprising single-chain gL/gH and ferritin (FIG. 11B incomparison to unconjugated sample in FIG. 11A) or comprising gp220 andferritin (FIG. 11D in comparison to unconjugated sample in FIG. 11C) didnot disrupt nanoparticle assembly.

Antibody responses were assayed by ELISA following immunization with 1μg of nanoparticles comprising single-chain gL/gH (gL_gH_C5 NP, FIGS.12A and 12B) or nanoparticles comprising gp220 (FIGS. 13A and 13B).Nanoparticles were in combination with 1 μg of naked ferritin and wereunconjugated or conjugated to SM7/8a. Unconjugated nanoparticles wereadministered with or without admixed AF03 adjuvant. Each mouse received100 μL of the nanoparticle composition as described above. For micereceiving AF03 adjuvant, a 1:1 volume of AF03 was mixed with thenanoparticles. BALB/c mice (n=5/group) were immunized twice with a3-week interval between doses. A bleed was taken for ELISA analysis atweek 5. The most robust ELISA responses were seen for nanoparticlesadministered in the AF03 adjuvant. Conjugation to SM7/8a produced a morerobust ELISA response compared to unconjugated nanoparticles withoutadjuvant.

The effect of coadministration of 1 μg each of gL_gH_C5 nanoparticlesconjugated to SM7/8a and gp220 nanoparticles conjugated to SM7/8a wasalso assessed, as compared to single administration of eithernanoparticle accompanied by naked ferritin nanoparticles in FIGS.14A-14B and 15A-15B. No interference was observed on the immune responseto either single-chain gL/gH (FIGS. 14A-14B, without and with AF03,respectively) or gp220 (FIGS. 15A-15B, without and with AF03,respectively).

4. Long-Term Immunogenicity Studies

Studies were performed to assess immunogenicity at 3 months after dosingwith nanoparticles comprising single-chain gL/gH (gL/gH_C5, SEQ ID NO:19). BALB/c mice (n=5/group) were immunized twice with a 3-week intervalbetween doses. Naked ferritin (i.e., ferritin not conjugated to anypolypeptide or adjuvant) was administered at 1 μg with the 1 μgnanoparticles comprising single-chain gL/gH, and the nanoparticles wereformulated in the presence or absence of admixed AF03 adjuvant. A bleedwas taken for ELISA analysis at week 13. For mice receiving AF03adjuvant, a 1:1 volume of AF03 was mixed with the nanoparticlecomposition. Each mouse received 100 μL of the nanoparticle compositiondescribed above. Some mice received nanoparticles comprisingsingle-chain gL/gH in which the ferritin was conjugated to SM7/8a(“7/8a” in FIGS. 16-17 ).

As shown in FIG. 16 , nanoparticles comprising single-chain gL/gHconjugated to SM7/8a produced the greatest immune response whenformulated in AF03. A robust immune response was also seen for thesenanoparticles without AF03.

A parallel experiment was performed using gp220 nanoparticles (SEQ IDNO: 1) (with or without conjugation to SM7/8a) in place of thenanoparticles comprising single-chain gL/gH. Similar results were seenfor these nanoparticles, wherein the formulation including admixed AF03produced the most robust response, and a robust immune response was alsoseen for these nanoparticles without AF03 (FIG. 17 ).

The immune response elicited by a bivalent composition comprisingnanoparticles comprising single-chain gL/gH (gL/gH_C5; SEQ ID NO: 19)and nanoparticles comprising gp220 (SEQ ID NO: 1) was assessed. BALB/cmice (n=5/group) were immunized with a 3-week interval between doses.100 μL of the nanoparticle composition containing 1 μg of eachnanoparticle was administered. For mice receiving AF03 adjuvant, a 1:1volume of AF03 was mixed with vaccine. A terminal week 13 bleed wastaken for ELISA analysis. For immune responses against both single-chaingL/gH (FIG. 18 ) and gp220 (FIG. 19 ), no interference was seen due toadministration of the nanoparticles in combination, as compared toadministration of either nanoparticle in combination with nakedferritin.

Further experiments with the gL/gH_C5 nanoparticle (SEQ ID NO: 19)confirmed that long-term immune responses were seen when thenanoparticle was conjugated to SM7/8a (7/8a) or when the nanoparticlewas formulated in AF03 (FIG. 21 ). BALB/c mice (n=5/group) wereimmunized with a 3-week interval between doses. 100 μL of thenanoparticle composition containing 1 μg of nanoparticles wasadministered. For mice receiving AF03 adjuvant, a 1:1 volume of AF03 wasmixed with vaccine. Week 2 (Prime), 5 (Boost), and 13 (Terminal) bleedswere taken for ELISA analysis. A parallel experiment was performed usinggp220 nanoparticles (SEQ ID NO: 1) and a similar long-term response wasalso seen for gp220 nanoparticles (FIG. 22 ).

A different nanoparticle comprising single-chain gL/gH (gL_gH_C7: SEQ IDNO: 20) was also assessed. The gL_gH_C7 construct comprises a flexiblelinker between the gH polypeptide and the ferritin with a cysteine as aconjugation site for an immune-stimulatory moiety. The linker may beused with a ferritin lacking a surface-exposed cysteine (as shown in SEQID NO: 20). SM7/8a was conjugated to gL_gH_C7 by reducing the proteinusing 2 mM TCEP and then oxidizing by adding 1×PBS and using a 100 kDmicrospin column to remove TCEP. The SM7/8a was then incubated with thegL/gH nanoparticle. Following conjugation, excess SM7/8a was removedfrom the reaction via a 100 kD microspin column.

Mice received 1 μg of these gL/gH nanoparticles, either conjugated to7/8a or unconjugated, plus 1 μg of naked ferritin. 100 μL of thenanoparticle composition containing 1 μg of nanoparticles wasadministered. BALB/c mice (n=5/group) were immunized with a 3-weekinterval between doses. For mice receiving AF03 adjuvant, a 1:1 volumeof AF03 was mixed with the nanoparticle composition. Week 2 (prime), 5(booster), and 13 (terminal) bleeds were taken for ELISA analysis. Thesenanoparticles elicited immune responses when formulated in AF03 or whenconjugated to SM7/8a as measured by ELISA endpoint titer at prime bleed(FIG. 20A). Similar results were seen with booster bleed (FIG. 20C) orterminal bleed (FIG. 20D) samples. These nanoparticles were alsoconjugated to a CpG oligodeoxynucleotide, and administered in the sameway. Results for the CpG conjugate were similar to unconjugatednanoparticles (FIG. 20B) at week 5.

5. Characterization of Nanoparticles Comprising Trichoplusia ni Ferritin

Nanoparticles were also developed comprising Trichoplusia ni ferritinand gp220 and/or gL/gH polypeptides. Trichoplusia ni ferritinnanoparticles contain heavy and light chains self-assembled at a 1:1ratio. It was found that combining one non-ferritin polypeptide with thelight chain and another non-ferritin polypeptide on the heavy chainallowed presentation of two distinct polypeptides on the surface ofindividual nanoparticles. Thus, for example, a self-assembledTrichoplusia ni ferritin nanoparticle could present both gp220 andgL/gH.

A Trichoplusia ni ferritin nanoparticle was produced and purified withthe heavy chain fused to either gp220 (SEQ ID NO: 24) or single-chaingL/gH (SEQ ID NO: 25) and the light chain fused to either gp220 (SEQ IDNO: 26) or single-chain gL/gH (SEQ ID NO: 27) (constructs illustrated inFIG. 23B and visualized by Coomassie gel staining in FIG. 23A, showingthe expected increase in molecular weight relative to light and heavychains alone). The combination of a light chain and a heavy chain fusedto gL/gH and gp220, respectively or vice versa, generated an individualmultivalent nanoparticle that can present two different EBVpolypeptides.

Two T. ni ferritin nanoparticles with either only gp220 in both theheavy and light chains (as shown in FIG. 24E) or gp220 in the heavychain and gH_gL in the light chain (as shown in FIG. 25E) were alsoproduced. The purification followed two steps: The first purificationstep was an ion exchange chromatographic step (Q column, see FIG. 24Awith Coomassie results in FIG. 24C and FIG. 25A with Coomassie resultsshown in FIG. 25C). This step was followed by size exclusionchromatography (see FIG. 24B with Coomassie results in FIGS. 24D and 25Bwith Coomassie results in FIG. 25D).

Nanoparticles comprising Trichoplusia ni light and heavy chain fused togp220 (SEQ ID NOs: 24 and 26; illustrated in FIG. 26B) showed a profileconsistent with formation of a nanoparticle comprising the heterologousgp220 polypeptide, based on Coomassie staining (FIG. 26A), an increasein DLS radius (FIG. 26D) relative to naked T. ni ferritin (FIG. 27C),and EM analysis (FIG. 26C) in which additional peripheral density aroundthe nanoparticle core appeared relative to the naked nanoparticles (FIG.27B). Similar results indicative of the presence of heterologous gL/gHand gp220 polypeptides in the nanoparticles were seen for SEQ ID NOs: 24and 27 (Trichoplusia ni light chains with a gL/gH polypeptide and heavychains with a gp220 polypeptide; see FIGS. 26E-26H for visualization byCoomassie staining, an illustration of the construct, an electronmicrograph, and characterization by DLS, respectively). For comparison,FIGS. 27A-27C show Coomassie staining (FIG. 27A), DLS radius (FIG. 27B),and EM analysis (FIG. 27C) for naked T. ni ferritin (i.e., notconjugated to any polypeptide).

Thus, use of T. ni ferritin allows presentation of 2 polypeptides onindividual nanoparticles.

6. gH/gL/gp42 Constructs

A cartoon of a single-chain construct of gH/gL/gp42 fused to ferritin(as in each of SEQ ID NOs: 227-231 and 241-242) is shown in FIG. 35A.The fusion between each protein is via a flexible amino acid linker or arigid amino acid linker. The single-chain gH/gL/gp42 molecule provides a1:1:1 ratio of heterotrimer formation on the nanoparticle.

The crystal structure of a gH/gL/gp42 His-tagged fusion (SEQ ID NO: 226)has been solved to show that the single-chain gH/gL/gp42 can adopt aheterotrimer conformation similar to wild-type gH, gL, and gp42 proteinsfound in nature (FIGS. 34 and 35B). In FIGS. 34 and 35B, Gp42 (in darkgray and indicated with arrows in FIG. 34 ) interacts with the gH/gLheterodimer. FIG. 35C is a model of how this single-chain gH/gL/gp42heterotrimer fused to ferritin is displayed on a nanoparticle. There aretwenty-four copies of the single-chain gH/gL/gp42 that will be displayedon a single nanoparticle.

A gH/gL/gp42 NP construct (SEQ ID NO: 227) was expressed in 293 expicells and purified (FIG. 28A). gH/gL/gp42 NP purified from CHO pools hada dynamic light scattering radius of around 26.2 nm (FIG. 28B).

The immune responses elicited by a monovalent (gH/gL/gp42 NP+nakedferritin nanoparticle) or bivalent (gH/gL/gp42 NP+gp220 NP) compositionwere assessed. The gH/gL/gp42 NP had the sequence of SEQ ID NO: 227 andthe gp220 NP had the sequence of SEQ ID NO: 1. BALB/c mice (n=5/group)were immunized with a 3-week interval between doses. 100 μL of thenanoparticle composition containing 1 μg of each nanoparticle wasadministered with an AF03 adjuvant (1:1 volume of AF03 mixed withvaccine). The boost indicates the sera collected at week 5 after thesecond immunization. EBV viral neutralizing assay analysis in B cells(FIG. 29A) and in epithelial cells (FIG. 29B) was done using seracollected at week 5 from the mice. No interference was seen due toadministration of the nanoparticles in bivalent formulation, as comparedto administration of the monovalent form (gH/gL/gp42 with nakedferritin).

Bivalent immunization of ferrets was performed using compositionscomprising single-chain gL/gH nanoparticles (gL_gH_C137A_bfpFerrNanoparticle N19Q/C31S/S111C [SEQ ID NO: 22]) and gp220 nanoparticles(SEQ ID NO: 1) in the presence of adjuvant AF03 (FIGS. 30A-30B) orgL/gH/gp42 NP (SEQ ID NO: 227) and gp220 nanoparticles (SEQ ID NO: 1) inthe presence of adjuvant AF03 (FIGS. 30C-30E). Inj. 1=injection one(sera collected from 6 ferrets at week 2 post Inj. 1). Inj. 2=injection2 (sera collected from 6 ferrets at week 2 post Inj. 2). An ELISAbinding assay measured endpoint binding titers against the antigensindicated in FIGS. 30A-30E. FIGS. 30F-G shows an EBV viral neutralizingassay (in B cells and epithelial cells, respectively) of sera fromferrets receiving bivalent vaccination of gL/gH/gp42 NP (SEQ ID NO: 227)and gp220 nanoparticles (SEQ ID NO: 1) in the presence of adjuvant AF03.Prime=Inj. 1 and Boost=Inj. 2.

gH/gL/gp42_NP_C12 (SEQ ID NO: 228) was expressed and purified usingSuperose 6 size exclusion chromatography (FIG. 31A). A dynamic lightscattering analysis of the sample in FIG. 31A showed a particle sizeradius of 20.6 nm (FIG. 31B).

gH/gL/gp42_NP_C13 (SEQ ID NO: 229) was expressed and purified usingSuperose 6 size exclusion chromatography (FIG. 32A). A dynamic lightscattering analysis of the sample in FIG. 32A showed a particle sizeradius of 17.1 nm (FIG. 32B).

gH/gL/gp42_NP_C14 (SEQ ID NO: 230) was expressed and purified usingSuperose 6 size exclusion chromatography (FIG. 33A). A dynamic lightscattering analysis of the sample in FIG. 33A showed a particle sizeradius of 16.9 nm (FIG. 33B).

FIG. 35D shows the purification of SEQ ID NO: 227 after expression in293Expi cells. A denaturing SDS coomassie gel shows the gH/gL/gp42 fusedto ferritin to be above 150 kD with glycosylation. Negative stainelectron microscopy analysis of the purified product shows thesingle-chain gH/gL/gp42 fused to ferritin can successfully formnanoparticles displaying the gH/gL/gp42 antigens on the surface (FIG.35E). Through temperature, oxidation, and/or deamidation stress test ondays 0, 3, 7, or 14, potential labile sequences have been identified viasequence analysis or mass spectrometry for the single-chain gH/gL/gp42nanoparticle of SEQ ID NO: 227. To improve vaccine stability,expression, and/or immunogenicity of this vaccine construct,conservative amino acid substitution mutations will be made to SEQ IDNO: 227 in different combinations, particularly at the sites listed inTable 1. Conservative amino acid mutations at the respective location inthe particular gene will also be tested in SEQ ID NOs: 228-230, whichdiffer from SEQ ID NOs. 227 only by the linker sequence that fuses theC-terminus of gp42 with the N-terminus of the ferritin sequence.

1.-39. (canceled)
 40. A single-chain Epstein Barr Virus (EBV)polypeptide comprising an EBV gL polypeptide, an EBV gH polypeptide, anEBV gp42 polypeptide, and a ferritin, wherein a linker having a lengthof at least 40 amino acids and less than or equal to 50 amino acidsseparates the EBV gL polypeptide and the EBV gH polypeptide, and whereinthe single-chain EBV polypeptide comprises a sequence having at least90% sequence identity to any one of SEQ ID NOs: 227-231, or to any oneof SEQ ID NOs: 241-242 lacking the leader sequence.
 41. A single-chainEpstein Barr Virus (EBV) polypeptide comprising an EBV gL polypeptide,an EBV gH polypeptide, an EBV gp42 polypeptide, and a ferritin, whereina linker having a length of 15 to 60 amino acids separates the EBV gHpolypeptide and the EBV gp42 polypeptide, and wherein the single-chainEBV polypeptide comprises a sequence having at least 90% sequenceidentity to any one of SEQ ID NOs: 227-231, or to any one of SEQ ID NOs:241-242 lacking the leader sequence.
 42. The single-chain EBVpolypeptide of claim 40, wherein a linker having a length of 15 to 60amino acids separates the EBV gH polypeptide and the EBV gp42polypeptide.
 43. The single-chain EBV polypeptide of claim 42, whereinthe single-chain EBV polypeptide comprises a sequence having at least95% sequence identity to SEQ ID NO:242 lacking the leader sequence. 44.The single-chain EBV polypeptide of claim 40, wherein the single-chainEBV polypeptide comprises a sequence having at least 98% sequenceidentity to SEQ ID NO:242 lacking the leader sequence.
 45. Thesingle-chain EBV polypeptide of claim 42, wherein the single-chain EBVpolypeptide comprises a sequence having at least 99% sequence identityto SEQ ID NO: 242 lacking the leader sequence, and wherein the linkerthat separates the EBV gH polypeptide and the EBV gp42 polypeptide has alength of 30 to 50 amino acids.
 46. The single-chain EBV polypeptide ofclaim 40, wherein the single-chain EBV polypeptide comprises the aminoacid sequence of SEQ ID NO:242 lacking the leader sequence.
 47. Thesingle-chain EBV polypeptide of claim 40, wherein the single-chain EBVpolypeptide consists of the amino acid sequence of SEQ ID NO:242 lackingthe leader sequence.
 48. The single-chain EBV polypeptide of claim 40,wherein the single-chain EBV polypeptide consists of the amino acidsequence of SEQ ID NO:227 lacking the leader sequence.
 49. Thesingle-chain Epstein Barr Virus (EBV) polypeptide of claim 40, whereinthe EBV gL polypeptide comprises the amino acid sequence of amino acids31-145 of SEQ ID NO: 242, the EBV gH polypeptide comprises the aminoacid sequence of amino acids 192-853 of SEQ ID NO: 242, and the EBV gp42polypeptide comprises the amino acid sequence of amino acids 886-1068 ofSEQ ID NO:
 242. 50. The single-chain EBV polypeptide of claim 49,wherein the EBV gL polypeptide, the EBV gH polypeptide and the EBV gp42polypeptide are in N- to C-terminal orientation.
 51. The single-chainEBV polypeptide of claim 50, wherein a linker having a length of atleast 40 amino acids and less than or equal to 50 amino acids separatesthe EBV gL polypeptide and the EBV gH polypeptide.
 52. The single-chainEBV polypeptide of claim 50, wherein a linker having a length of 46 or47 amino acids separates the EBV gL polypeptide and the EBV gHpolypeptide.
 53. The single-chain EBV polypeptide of claim 50, wherein alinker having a length of 15 to 60 amino acids separates the EBV gHpolypeptide and the EBV gp42 polypeptide.
 54. The single-chain EBVpolypeptide of claim 51, wherein a linker having a length of 30 to 50amino acids separates the EBV gH polypeptide and the EBV gp42polypeptide.
 55. The single-chain EBV polypeptide of claim 53, whereinthe linker comprises one or more of glycine, asparagine, serine, andalanine.
 56. The single-chain EBV polypeptide of claim 54, wherein thelinker comprises one or more of glycine, asparagine, serine, andalanine.
 57. A single-chain EBV polypeptide comprising the amino acidsequence of amino acids 31-1156 of SEQ ID NO:
 242. 58. The single-chainEBV polypeptide of claim 57, further comprising a ferritin, wherein theamino acid sequence of amino acids 31-1156 of SEQ ID NO: 242 and theferritin are in N- to C-terminal orientation, and wherein the ferritinhas at least 90% sequence identity to the amino acid sequence of any oneof SEQ ID NOs: 201 to
 208. 59. The single-chain EBV polypeptide of claim58, wherein the ferritin has at least 95% sequence identity to the aminoacid sequence of SEQ ID NO:
 206. 60. The single-chain EBV polypeptide ofclaim 58, wherein the ferritin has at least 97% sequence identity to theamino acid sequence of SEQ ID NO:
 206. 61. A composition comprising afirst single-chain EBV polypeptide of claim 58, and a secondsingle-chain EBV polypeptide comprising an EBV gp220 polypeptide and asecond ferritin.
 62. The composition of claim 61, wherein the gp220polypeptide has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:
 38. 63. The composition of claim 61, wherein thegp220 polypeptide has at least 99% sequence identity to the amino acidsequence of SEQ ID NO:
 38. 64. The composition of claim 61, wherein thegp220 polypeptide consists of the amino acid sequence of SEQ ID NO: 38.65. The composition of claim 61, wherein the second ferritin has atleast 95% sequence identity to the amino acid sequence of SEQ ID NO:206.
 66. The composition of claim 61, wherein the second single-chainEBV polypeptide comprises the amino acid sequence of SEQ ID NO: 1lacking the leader sequence.
 67. A composition comprising (a) a firstsingle-chain EBV polypeptide consisting of the amino acid sequence ofamino acids 31-1156 of SEQ ID NO: 242 and a first ferritin having anamino acid sequence at least 97% identical to SEQ ID NO: 206, whereinthe amino acid sequence of amino acids 31-1156 of SEQ ID NO: 242 and thefirst ferritin are in N- to C-terminal orientation; and (b) a secondsingle-chain EBV polypeptide consisting of the amino acid sequence ofSEQ ID NO: 1 lacking the leader sequence.
 68. The composition of claim67, further comprising an adjuvant.
 69. A composition comprising asingle-chain EBV polypeptide consisting of the amino acid sequence ofSEQ ID NO: 1 lacking the leader sequence.